Human antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof

ABSTRACT

The present disclosure provides isolated monoclonal antibodies that specifically bind to LAG-3 with high affinity, particularly human monoclonal antibodies. Preferably, the antibodies bind human LAG-3. In certain embodiments, the antibodies bind both human and monkey LAG-3 but do not bind mouse LAG-3. The invention provides anti-LAG-3 antibodies that can inhibit the binding of LAG-3 to MHC Class II molecules and that can stimulate antigen-specific T cell responses. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells and methods for expressing the antibodies of the invention are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the invention are also provided. This disclosure also provides methods for detecting LAG-3, as well as methods for treating stimulating immune responses using an anti-LAG-3 antibody of the invention. Combination therapy, in which an anti-LAG-3 antibody is co-administered with at least one additional immunostimulatory antibody, is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/730,363, filed Oct. 11, 2017, and U.S. Pat. No. 10,344,089, which isa divisional of U.S. application Ser. No. 13/058,492, 371(c) date Feb.10, 2011, which is the National Stage of International Application No.PCT/US2009/053405, filed Aug. 11, 2009, which claims the benefit under35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/188,548, filedAug. 11, 2008; the disclosures of each of which are incorporated hereinby reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:3338_0770003_Seqlisting_ST25.txt; Size: 50,533 bytes; and Date ofCreation: May 22, 2019) is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Lymphocyte Activation Gene-3, or LAG-3 (also know as CD223), is a memberof the immunoglobulin supergene family and is structurally andgenetically related to CD4. LAG-3 is not expressed on resting peripheralblood lymphocytes but is expressed on activated T cells and NK cells.LAG-3 is a membrane protein encoded by a gene located on the distal partof the short arm of chromosome 12, near the CD4 gene, suggesting thatthe LAG-3 gene may have evolved through gene duplication (Triebel et al.(1990) J. Exp. Med. 171:1393-1405).

Similar to CD4, LAG-3 has been demonstrated to interact with MHC ClassII molecules but, unlike CD4, LAG-3 does not interact with the humanimmunodeficiency virus gp120 protein (Baixeras et al. (1992) J. Exp.Med. 176:327-337). Studies using a soluble LAG-3 immunoglobulin fusionprotein (sLAG-3Ig) demonstrated direct and specific binding of LAG-3 toMHC class II on the cell surface (Huard et al. (1996) Eur. J. Immunol.26:1180-1186).

In in vitro studies of antigen-specific T cell responses, the additionof anti-LAG-3 antibodies led to increased T cell proliferation, higherexpression of activation antigens such as CD25, and higherconcentrations of cytokines such as interferon-gamma and interleukin-4,supporting a role for the LAG−/MHC class II interaction indown-regulating antigen-dependent stimulation of CD4⁺ T lymphocytes(Huard et al. (1994) Eur. J. Immunol. 24:3216-3221). Theintra-cytoplasmic region of LAG-3 has been demonstrated to interact witha protein termed LAP, which is thought to be a signal transductionmolecule involved in the downregulation of the CD3/TCR activationpathway (Iouzalen et al. (2001) Eur. J. Immunol. 31:2885-2891).Furthermore, CD4⁺CD25⁺ regulatory T cells (T_(reg)) have been shown toexpress LAG-3 upon activation and antibodies to LAG-3 inhibitsuppression by induced T_(reg) cells, both in vitro and in vivo,suggesting that LAG-3 contributes to the suppressor activity of T_(reg)cells (Huang, C. et al. (2004) Immunity 21:503-513). Still further,LAG-3 has been shown to negatively regulate T cell homeostasis byregulatory T cells in both T cell-dependent and independent mechanisms(Workman, C. J. and Vignali, D. A. (2005) J. Immunol. 174:688-695).

In certain circumstances, LAG-3 also has been shown to haveimmunostimulatory effects. For example, LAG-3 transfected tumor cellstransplanted into syngeneic mice showed marked growth reduction orcomplete regression as compared to untransfected tumor cells, suggestingthat LAG-3 expression on the tumor cells stimulated an anti-tumorresponse by triggering antigen presenting cells via MHC class IImolecules (Prigent et al. (1999) Eur. J. Immunol. 29:3867-3876).Additionally, soluble LAG-3 Ig fusion protein has been shown tostimulate both humoral and cellular immune responses when administeredto mice together with an antigen, indicating that soluble LAG-3Ig canfunction as a vaccine adjuvant (El Mir and Triebel (2000) J. Immunol.164:5583-5589). Furthermore, soluble human LAG-3Ig has been shown toamplify the in vitro generation of type I tumor-specific immunity(Casati et al. (2006) Cancer Res. 66:4450-4460). The functional activityof LAG-3 is reviewed further in Triebel (2003) Trends Immunol.24:619-622. In view of the above, additional agents for modulating theactivity of LAG-3 are of interest.

SUMMARY

The present disclosure provides isolated monoclonal antibodies, inparticular human monoclonal antibodies, that specifically bind LAG-3 andthat have desirable functional properties. These properties include highaffinity binding to human LAG-3, binding to human and monkey LAG-3(e.g., cynomolgus and/or rhesus monkey LAG-3) but not to mouse LAG-3,the ability to inhibit binding of LAG-3 to major histocompatibility(MHC) Class II molecules and/or the ability to stimulateantigen-specific T cell responses. The antibodies of the invention canbe used, for example, to detect LAG-3 protein or to stimulateantigen-specific T cell responses, such as in a tumor-bearing subject ora virus-bearing subject.

In one aspect, the invention pertains to an isolated human monoclonalantibody, or an antigen-binding portion thereof, wherein the antibodybinds human LAG-3 and exhibits at least one of the following properties:

(a) binds monkey LAG-3;

(b) does not bind mouse LAG-3;

(c) inhibits binding of LAG-3 to major histocompatibility (MHC) class IImolecules; and

(d) stimulates an immune response.

Preferably, the antibody exhibits at least two of properties (a), (b),(c) and (d). More preferably, the antibody exhibits at least three ofproperties (a), (b), (c) and (d). Even more preferably, the antibodyexhibits all four of properties (a), (b), (c) and (d).

In a preferred embodiment, the antibody stimulates an antigen-specific Tcell response, such as interleukin-2 (IL-2) production in anantigen-specific T cell response. In other embodiments, the antibodystimulates an immune response such as an anti-tumor response (e.g.,inhibits tumor growth in an in vivo tumor graft model) or an autoimmuneresponse (e.g., promotes the development of diabetes in NOD mice). Inanother preferred embodiment, the antibody binds an epitope of humanLAG-3 comprising the amino acid sequence PGHPLAPG (SEQ ID NO: 76). Inyet another preferred embodiment, the antibody binds an epitope of humanLAG-3 comprising the amino acid sequence HPAAPSSW (SEQ ID NO: 77) orPAAPSSWG (SEQ ID NO: 78). In still other embodiments, the antibody bindsto human LAG-3 with a K_(D) of 1×10⁻⁷ M or less, or binds to human LAG-3with a K_(D) of 1×10⁻⁸ M or less, or binds to human LAG-3 with a K_(D)of 5×10⁻⁹ M or less, or binds to human LAG-3 with a K_(D) of 1×10⁻⁹ M orless. In one embodiment, the antibody stains pituitary tissue byimmunohistochemistry, whereas in another embodiment, the antibody doesnot stain pituitary tissue by immunohistochemistry.

In another aspect, the invention pertains to an isolated humanmonoclonal antibody, or antigen binding portion thereof, wherein theantibody cross-competes for binding to human LAG-3 with a referenceantibody, wherein the reference antibody comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 37 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 43;

(b) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 38 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 44;

(c) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 39 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 45;

(d) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 40 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 46;

(e) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 41 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 47; or

(f) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 42 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 48.

In a preferred embodiment, the reference antibody comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:37 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 43. In another preferred embodiment, the referenceantibody comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 38 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 44. In anotherpreferred embodiment, the reference antibody comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 39 anda light chain variable region comprising the amino acid sequence of SEQID NO: 45. In another preferred embodiment, the reference antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 40 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 46. In another preferredembodiment, the reference antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 41 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:47. In another preferred embodiment, the reference antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 42 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 48.

In another aspect, the invention pertains to an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a heavychain variable region that is the product of or derived from a humanV_(H) 3-20 gene, a human V_(H) 4-34 gene, a human V_(H) 3-33 gene or ahuman V_(H) 1-24 gene, wherein the antibody specifically binds humanLAG-3. In another aspect, the invention pertains to an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising alight chain variable region that is the product of or derived from ahuman V_(K) L18 gene, a human V_(K) L6 gene or a human V_(K) A27 gene,wherein the antibody specifically binds human LAG-3. In a preferredembodiment, the invention provides an isolated monoclonal antibody, oran antigen-binding portion thereof, comprising:

(a) a heavy chain variable region that is the product of or derived froma human V_(H) 4-34 gene and a light chain variable region that is theproduct of or derived from a human V_(K) L6 gene;

(b) a heavy chain variable region that is the product of or derived froma human V_(H) 3-33 gene and a light chain variable region that is theproduct of or derived from a human V_(K) A27 gene;

(c) a heavy chain variable region that is the product of or derived froma human V_(H) 3-20 gene and a light chain variable region that is theproduct of or derived from a human V_(K) L18 gene;

(d) a heavy chain variable region that is the product of or derived froma human V_(H) 1-24 gene and a light chain variable region that is theproduct of or derived from a human V_(K) L6 gene; or

(e) a heavy chain variable region that is the product of or derived froma human V_(H) 3-33 gene and a light chain variable region that is theproduct of or derived from a human V_(K) L6 gene;

wherein the antibody specifically binds human LAG-3.

In another aspect, the invention pertains to an isolated monoclonalantibody, or antigen binding portion thereof, comprising:

(a) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 37-42;

(b) a light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 43-48;

wherein the antibody specifically binds human LAG-3.

A preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 37; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 43.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 38; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 44.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 39; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 45.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 40; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 46.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 41; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 47.

Another preferred combination comprises:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 42; and

(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 48.

The antibodies of the invention can be, for example, full-lengthantibodies, for example of an IgG1, IgG2 or IgG4 isotype. In a preferredembodiment, the antibody is an IgG4 isotype. In another preferredembodiment, the antibody is an IgG4 isotype having a serine to prolinemutation in the heavy chain constant region hinge region (at a positioncorresponding to position 241 as described in Angal et al. (1993) Mol.Immunol. 30:105-108), such that inter-heavy chain disulfide bridgeheterogeneity is reduced or abolished. Alternatively, the antibodies canbe antibody fragments, such as Fab, Fab′ or Fab′2 fragments, or singlechain antibodies.

This disclosure also provides an immunoconjugate comprising an antibodyof the invention, or antigen-binding portion thereof, linked to atherapeutic agent, e.g., a cytotoxin or a radioactive isotope. Thisdisclosure also provides a bispecific molecule comprising an antibody,or antigen-binding portion thereof, of the invention, linked to a secondfunctional moiety having a different binding specificity than saidantibody, or antigen binding portion thereof.

Compositions comprising an antibody, or antigen-binding portion thereof,or immunoconjugate or bispecific molecule of the invention and apharmaceutically acceptable carrier are also provided.

Nucleic acid molecules encoding the antibodies, or antigen-bindingportions thereof, of the invention are also encompassed by thisdisclosure, as well as expression vectors comprising such nucleic acidsand host cells comprising such expression vectors. Methods for preparinganti-LAG-3 antibodies using the host cells comprising such expressionvectors are also provided and can include the steps of (i) expressingthe antibody in the host cell and (ii) isolating the antibody from thehost cell.

In another aspect, the invention pertains to methods of stimulatingimmune responses using the anti-LAG-3 antibodies of the invention. Forexample, in one embodiment, the invention provides a method ofstimulating an antigen-specific T cell response comprising contactingsaid T cell with an antibody of the invention such that anantigen-specific T cell response is stimulated. In a preferredembodiment, interleukin-2 production by the antigen-specific T cell isstimulated. In another embodiment, the invention provides a method ofstimulating an immune response (e.g., an antigen-specific T cellresponse) in a subject comprising administering an antibody of theinvention to the subject such that an immune response (e.g., anantigen-specific T cell response) in the subject is stimulated. In apreferred embodiment, the subject is a tumor-bearing subject and animmune response against the tumor is stimulated. In another preferredembodiment, the subject is a virus-bearing subject and an immuneresponse against the virus is stimulated.

In yet another aspect, the invention provides a method for inhibitinggrowth of tumor cells in a subject comprising administering to thesubject an antibody of the invention such that growth of the tumor isinhibited in the subject. In still another aspect, the inventionprovides a method for treating viral infection in a subject comprisingadministering to the subject an antibody of the invention such that theviral infection is treated in the subject.

In yet another aspect, the invention provides a method for stimulatingan immune response in a subject comprising administering to the subjectan anti-LAG-3 antibody and at least one additional immunostimulatoryantibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody and/oran anti-CTLA-4 antibody, such that an immune response is stimulated inthe subject, for example to inhibit tumor growth or to stimulate ananti-viral response. In one embodiment, the subject is administered ananti-LAG-3 antibody and an anti-PD-1 antibody. In another embodiment,the subject is administered an anti-LAG-3 antibody and an anti-PD-L1antibody. In yet another embodiment, the subject is administered ananti-LAG-3 antibody and an anti-CTLA-4 antibody. In one embodiment, theanti-LAG-3 antibody is a human antibody, such as an antibody of thedisclosure. Alternatively, the anti-LAG-3 antibody can be, for example,a chimeric or humanized antibody. In another embodiment, the at leastone additional immunostimulatory antibody (e.g., anti-PD-1, anti-PD-L1and/or anti-CTLA-4 antibody) is a human antibody. Alternatively, the atleast one additional immunostimulatory antibody can be, for example, achimeric or humanized antibody.

In yet another aspect, the invention pertains to a method for preparingan anti-LAG-3 antibody. The method comprises:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 sequence selected from the group consisting of SEQ IDNOs: 1-6, a CDR2 sequence selected from the group consisting of SEQ IDNOs: 7-12, and/or a CDR3 sequence selected from the group consisting ofSEQ ID NOs: 13-14, GGY and 16-18; and/or (ii) a light chain variableregion antibody sequence comprising a CDR1 sequence selected from thegroup consisting of SEQ ID NOs: 19-24, a CDR2 sequence selected from thegroup consisting of SEQ ID NOs: 25-30, and/or a CDR3 sequence selectedfrom the group consisting of SEQ ID NOs: 31-36;

(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples, which should notbe construed as limiting. The contents of all references, Genbankentries, patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 49) and amino acidsequence (SEQ ID NO: 37) of the heavy chain variable region of the 25F7human monoclonal antibody. The CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 7)and CDR3 (SEQ ID NO: 13) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 55) and amino acidsequence (SEQ ID NO: 43) of the kappa light chain variable region of the25F7 human monoclonal antibody. The CDR1 (SEQ ID NO: 19), CDR2 (SEQ IDNO: 25) and CDR3 (SEQ ID NO: 31) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 50) and amino acidsequence (SEQ ID NO: 38) of the heavy chain variable region of the 26H10human monoclonal antibody. The CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 8)and CDR3 (SEQ ID NO: 14) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 56) and amino acidsequence (SEQ ID NO: 44) of the kappa light chain variable region of the26H10 human monoclonal antibody. The CDR1 (SEQ ID NO: 20), CDR2 (SEQ IDNO: 26) and CDR3 (SEQ ID NO: 32) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 51) and amino acidsequence (SEQ ID NO: 39) of the heavy chain variable region of the 25E3human monoclonal antibody. The CDR1 (SEQ ID NO: 3), CDR2 (SEQ ID NO: 9)and CDR3 regions are delineated and the V, D and J germline derivationsare indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 57) and amino acidsequence (SEQ ID NO: 45) of the kappa light chain variable region of the25E3 human monoclonal antibody. The CDR1 (SEQ ID NO: 21), CDR2 (SEQ IDNO: 27) and CDR3 (SEQ ID NO: 33) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 52) and amino acidsequence (SEQ ID NO: 40) of the heavy chain variable region of the 8B7human monoclonal antibody. The CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 10)and CDR3 (SEQ ID NO: 16) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 58) and amino acidsequence (SEQ ID NO: 46) of the kappa light chain variable region of the8B7 human monoclonal antibody. The CDR1 (SEQ ID NO: 22), CDR2 (SEQ IDNO: 28) and CDR3 (SEQ ID NO: 34) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 5A shows the nucleotide sequence (SEQ ID NO: 53) and amino acidsequence (SEQ ID NO: 41) of the heavy chain variable region of the 11F2human monoclonal antibody. The CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 11)and CDR3 (SEQ ID NO: 17) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 5B shows the nucleotide sequence (SEQ ID NO: 59) and amino acidsequence (SEQ ID NO: 47) of the kappa light chain variable region of the11F2 human monoclonal antibody. The CDR1 (SEQ ID NO: 23), CDR2 (SEQ IDNO: 29) and CDR3 (SEQ ID NO: 35) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 6A shows the nucleotide sequence (SEQ ID NO: 54) and amino acidsequence (SEQ ID NO: 42) of the heavy chain variable region of the 17E5human monoclonal antibody. The CDR1 (SEQ ID NO: 6), CDR2 (SEQ ID NO: 12)and CDR3 (SEQ ID NO: 18) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 6B shows the nucleotide sequence (SEQ ID NO: 60) and amino acidsequence (SEQ ID NO: 48) of the kappa light chain variable region of the17E5 human monoclonal antibody. The CDR1 (SEQ ID NO: 24), CDR2 (SEQ IDNO: 30) and CDR3 (SEQ ID NO: 36) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 7 shows the alignment of the amino acid sequence of the heavy chainvariable regions of 25F7 (SEQ ID NO: 37) with the human germline V_(H)4-34 and JH5b amino acid sequences (SEQ ID NOS: 61 and 62,respectively).

FIG. 8 shows the alignment of the amino acid sequence of the light chainvariable region of 25F7 (SEQ ID NO: 43) with the human germline V_(k) L6and JK2 amino acid sequences (SEQ ID NOS: 63 and 64, respectively).

FIG. 9 shows the alignment of the amino acid sequence of the heavy chainvariable regions of 26H10 (SEQ ID NO: 38) with the human germline V_(H)3-33 and JH6B amino acid sequences (SEQ ID NOS: 65 and 66,respectively).

FIG. 10 shows the alignment of the amino acid sequence of the lightchain variable region of 26H10 (SEQ ID NO: 44) with the human germlineV_(k) A27 and JK3 amino acid sequences (SEQ ID NO: 67 and 68,respectively).

FIG. 11 shows the alignment of the amino acid sequence of the heavychain variable regions of 25E3 (SEQ ID NO: 39) with the human germlineV_(H) 3-20 and JH4b amino acid sequences (SEQ ID NOS: 69 and 70,respectively).

FIG. 12 shows the alignment of the amino acid sequence of the lightchain variable region of 25E3 (SEQ ID NO: 45) with the human germlineV_(k) L18 and JK2 amino acid sequences (SEQ ID NOS: 71 and 64,respectively).

FIG. 13 shows the alignment of the amino acid sequence of the heavychain variable regions of 8B7 (SEQ ID NO: 40) with the human germlineV_(H) 4-34 and JH5b amino acid sequences (SEQ ID NOS: 61 and 62,respectively).

FIG. 14 shows the alignment of the amino acid sequence of the lightchain variable region of 8B7 (SEQ ID NO: 46) with the human germlineV_(k) L6 and JK4 amino acid sequences (SEQ ID NOS: 63 and 72,respectively).

FIG. 15 shows the alignment of the amino acid sequence of the heavychain variable regions of 11F2 (SEQ ID NO: 41) with the human germlineV_(H) 1-24 and JH4b amino acid sequences (SEQ ID NOS: 73 and 70,respectively).

FIG. 16 shows the alignment of the amino acid sequence of the lightchain variable region of 11F2 (SEQ ID NO: 47) with the human germlineV_(k) L6 and JK1 amino acid sequences (SEQ ID NOS: 63 and 74,respectively).

FIG. 17 shows the alignment of the amino acid sequence of the heavychain variable regions of 17E5 (SEQ ID NO: 42) with the human germlineV_(H) 3-33 and 2-2 amino acid sequences (SEQ ID NOS: 65 and 70,respectively).

FIG. 18 shows the alignment of the amino acid sequence of the lightchain variable region of 17E5 (SEQ ID NO: 48) with the human germlineV_(k) L6 amino acid sequence (SEQ ID NOS: 63 and 75, respectively).

FIGS. 19A and B show the alignment of the protein sequence encoded bythe monkey LAG-3 cDNA clone pa23-5 (SEQ ID NO: 93) with the Genbankdeposited rhesus monkey LAG-3 protein sequence (SEQ ID NO: 94) (GenbankAccession No. XM_001108923). The extra loop peptide region andtransmembrane domain are underlined. The one amino acid differencebetween the two sequences (amino acid position 419) is highlighted inbold.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to isolated monoclonal antibodies,particularly human monoclonal antibodies, which bind to human LAG-3 andthat have desirable functional properties. In certain embodiments, theantibodies of the invention are derived from particular heavy and lightchain germline sequences and/or comprise particular structural featuressuch as CDR regions comprising particular amino acid sequences. Thisdisclosure provides isolated antibodies, methods of making suchantibodies, immunoconjugates and bispecific molecules comprising suchantibodies and pharmaceutical compositions containing the antibodies,immunoconjugates or bispecific molecules of the invention. Thisdisclosure also relates to methods of using the antibodies, such as todetect LAG-3 protein, as well as to methods of using the anti-LAG-3antibodies of the invention to stimulate immune responses, alone or incombination with other immunostimulatory antibodies. Accordingly, thisdisclosure also provides methods of using the anti-LAG-3 antibodies ofthe invention to, for example, inhibit tumor growth or treat viralinfection.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “LAG-3” refers to Lymphocyte Activation Gene-3. The term“LAG-3” includes variants, isoforms, homologs, orthologs and paralogs.For example, antibodies specific for a human LAG-3 protein may, incertain cases, cross-react with a LAG-3 protein from a species otherthan human. In other embodiments, the antibodies specific for a humanLAG-3 protein may be completely specific for the human LAG-3 protein andmay not exhibit species or other types of cross-reactivity, or maycross-react with LAG-3 from certain other species but not all otherspecies (e.g., cross-react with monkey LAG-3 but not mouse LAG-3). Theterm “human LAG-3” refers to human sequence LAG-3, such as the completeamino acid sequence of human LAG-3 having Genbank Accession No.NP_002277. The term “mouse LAG-3” refers to mouse sequence LAG-3, suchas the complete amino acid sequence of mouse LAG-3 having GenbankAccession No. NP_032505. LAG-3 is also known in the art as, for example,CD223. The human LAG-3 sequence may differ from human LAG-3 of GenbankAccession No. NP_002277 by having, e.g., conserved mutations ormutations in non-conserved regions and the LAG-3 has substantially thesame biological function as the human LAG-3 of Genbank Accession No.NP_002277. For example, a biological function of human LAG-3 is havingan epitope in the extracellular domain of LAG-3 that is specificallybound by an antibody of the instant disclosure or a biological functionof human LAG-3 is binding to MHC Class II molecules.

The term “monkey LAG-3” is intended to encompass LAG-3 proteinsexpressed by Old World and New World monkeys, including but not limitedto cynomolgus monkey LAG-3 and rhesus monkey LAG-3. A representativeamino acid sequence for monkey LAG-3 is the rhesus monkey LAG-3 aminoacid sequence shown in FIG. 19 and SEQ ID NO: 85, which is alsodeposited as Genbank Accession No. XM_001108923. Another representativeamino acid sequence for monkey LAG-3 is the alternative rhesus monkeysequence of clone pa23-5 shown in FIG. 19 and SEQ ID NO: 84, isolated asdescribed in Example 3A, subsection 3. This alternative rhesus sequenceexhibits a single amino acid difference, at position 419, as compared tothe Genbank-deposited sequence.

A particular human LAG-3 sequence will generally be at least 90%identical in amino acids sequence to human LAG-3 of Genbank AccessionNo. NP_002277 and contains amino acid residues that identify the aminoacid sequence as being human when compared to LAG-3 amino acid sequencesof other species (e.g., murine). In certain cases, a human LAG-3 can beat least 95%, or even at least 96%, 97%, 98%, or 99% identical in aminoacid sequence to LAG-3 of Genbank Accession No. NP_002277. In certainembodiments, a human LAG-3 sequence will display no more than 10 aminoacid differences from the LAG-3 sequence of Genbank Accession No.NP_002277. In certain embodiments, the human LAG-3 can display no morethan 5, or even no more than 4, 3, 2, or 1 amino acid difference fromthe LAG-3 sequence of Genbank Accession No. NP_002277. Percent identitycan be determined as described herein.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

An “antigen-specific T cell response” refers to responses by a T cellthat result from stimulation of the T cell with the antigen for whichthe T cell is specific. Non-limiting examples of responses by a T cellupon antigen-specific stimulation include proliferation and cytokineproduction (e.g., IL-2 production).

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. Whole antibodies are glycoproteins comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as V_(H)) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, C_(H)1,C_(H)2 and C_(H)3. Each light chain is comprised of a light chainvariable region (abbreviated herein as V_(L)) and a light chain constantregion. The light chain constant region is comprised of one domain,C_(L). The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies canmediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (Clq) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., a LAG-3 protein). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fab′fragment, which is essentially an Fab with part of the hinge region(see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993); (iv) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (v) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vii) an isolated complementaritydetermining region (CDR); and (viii) a nanobody, a heavy chain variableregion containing a single variable domain and two constant domains.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds a LAG-3 protein is substantially free of antibodies thatspecifically bind antigens other than LAG-3 proteins). An isolatedantibody that specifically binds a human LAG-3 protein may, however,have cross-reactivity to other antigens, such as LAG-3 proteins fromother species. Moreover, an isolated antibody can be substantially freeof other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences.Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention can include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity, which have variable regions in which boththe framework and CDR regions are derived from human germlineimmunoglobulin sequences. In one embodiment, the human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic nonhuman animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

The term “isotype” refers to the antibody class (e.g., IgM or IgGl) thatis encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications can be made withinthe human framework sequences.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

As used herein, an antibody that “specifically binds human LAG-3” isintended to refer to an antibody that binds to human LAG-3 protein (andpossibly a LAG-3 protein from one or more non-human species) but doesnot substantially bind to non-LAG-3 proteins. Preferably, the antibodybinds to a human LAG-3 protein with “high affinity”, namely with a K_(D)of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ M or less, more preferably3×10⁻⁸ M or less, more preferably 1×10⁻⁸ M or less, more preferably5×10⁻⁹ M or less or even more preferably 1×10⁻⁹ M or less.

The term “does not substantially bind” to a protein or cells, as usedherein, means does not bind or does not bind with a high affinity to theprotein or cells, i.e. binds to the protein or cells with a K_(D) of1×10⁻⁶ M or more, more preferably 1×10⁻⁵ M or more, more preferably1×10⁻⁴ M or more, more preferably 1×10⁻³ M or more, even more preferably1×10⁻² M or more.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D),” as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system.

The term “high affinity” for an IgG antibody refers to an antibodyhaving a K_(D) of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ M or less,even more preferably 1×10⁻⁸ M or less, even more preferably 5×10⁻⁹ M orless and even more preferably 1×10⁻⁹ M or less for a target antigen.However, “high affinity” binding can vary for other antibody isotypes.For example, “high affinity” binding for an IgM isotype refers to anantibody having a K_(D) of 10⁻⁶ M or less, more preferably 10⁻⁷ M orless, even more preferably 10⁻⁸ M or less.

The term “subject” includes any human or nonhuman animal. The term“nonhuman animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, cows,horses, chickens, amphibians, and reptiles, although mammals arepreferred, such as non-human primates, sheep, dogs, cats, cows andhorses.

Various aspects of the invention are described in further detail in thefollowing subsections.

Anti-LAG-3 Antibodies Having Particular Functional Properties

The antibodies of the invention are characterized by particularfunctional features or properties of the antibodies. For example, theantibodies specifically bind to human LAG-3 and may bind to LAG-3 fromcertain other species, e.g., monkey LAG-3 (e.g., cynomolgus monkey,rhesus monkey), but do not substantially bind to LAG-3 from certainother species, e.g., mouse LAG-3. Preferably, an antibody of theinvention binds to human LAG-3 with high affinity.

The ability of the antibody to stimulate an immune response, such as anantigen-specific T cell response, can be indicated by, for example, theability of the antibody to stimulate interleukin-2 (IL-2) production inan antigen-specific T cell response. In certain embodiments, an antibodyof the invention binds to human LAG-3 and exhibits an ability tostimulate an antigen-specific T cell response. In other embodiments, anantibody of the invention binds to human LAG-3 but does not exhibit anability to stimulate an antigen-specific T cell response. Other means bywhich to evaluate the ability of the antibody to stimulate an immuneresponse include the ability of the antibody to inhibit tumor growth,such as in an in vivo tumor graft model (see, e.g., Example 6) or theability of the antibody to stimulate an autoimmune response, such as theability to promote the development of an autoimmune disease in anautoimmune model, such as the ability to promote the development ofdiabetes in the NOD mouse model (see, e.g., Example 7).

The binding of an antibody of the invention to LAG-3 can be assessedusing one ore more techniques well established in the art. For example,in a preferred embodiment, an antibody can be tested by a flow cytometryassay in which the antibody is reacted with a cell line that expresseshuman LAG-3, such as CHO cells that have been transfected to expressLAG-3 (e.g., human LAG-3, or monkey LAG-3 (e.g., rhesus or cynomolgusmonkey) or mouse LAG-3) on their cell surface (see, e.g., Example 3A fora suitable assay). Other suitable cells for use in flow cytometry assaysinclude anti-CD3-stimulated CD4⁺ activated T cells, which express nativeLAG-3. Additionally or alternatively, the binding of the antibody,including the binding kinetics (e.g., K_(D) value) can be tested inBIAcore binding assays (see, e.g., Example 3B for suitable assays).Still other suitable binding assays include ELISA assays, for exampleusing a recombinant LAG-3 protein (see, e.g., Example 1 for a suitableassay).

Preferably, an antibody of the invention binds to a LAG-3 protein with aK_(D) of 5×10⁻⁸ M or less, binds to a LAG-3 protein with a K_(D) of2×10⁻⁸ M or less, binds to a LAG-3 protein with a K_(D) of 5×10⁻⁹ M orless, binds to a LAG-3 protein with a K_(D) of 4×10⁻⁹ M or less, bindsto a LAG-3 protein with a K_(D) of 3×10⁻⁹ M or less, binds to a LAG-3protein with a K_(D) of 2×10⁻⁹ M or less, binds to a LAG-3 protein witha K_(D) of 1×10⁻⁹ M or less, binds to a LAG-3 protein with a K_(D) of5×10⁻¹⁰ M or less, or binds to a LAG-3 protein with a K_(D) of 1×10⁻¹⁰ Mor less.

Typically, an antibody of the invention binds to LAG-3 in lymphoidtissues, such as tonsil, spleen or thymus, which can be detected byimmunohistochemistry. Additionally, as described further in Example 8,certain anti-LAG-3 antibodies of the invention stain pituitary tissue(e.g., are retained in the pituitary) as measured byimmunohistochemistry, whereas other anti-LAG-3 antibodies of theinvention do not stain pituitary tissue (e.g., are not retained in thepituitary) as measured by immunohistochemistry. Thus, in one embodiment,the invention provides a human anti-LAG-3 antibody that stains pituitarytissue by immunohistochemistry, whereas in another embodiment, theinvention provides a human anti-LAG-3 antibody that does not stainpituitary tissue by immunohistochemistry.

Preferred antibodies of the invention are human monoclonal antibodies.Additionally or alternatively, the antibodies can be, for example,chimeric or humanized monoclonal antibodies.

Monoclonal Antibodies 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5

Preferred antibodies of the invention are the human monoclonalantibodies 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5 isolated andstructurally characterized as described in Examples 1 and 2. The V_(H)amino acid sequences of 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5 are shownin SEQ ID NOs: 37-42, respectively. The V_(K) amino acid sequences of25F7, 26H10, 25E3, 8B7, 11F2 and 17E5 are shown in SEQ ID NOs: 43-48,respectively.

Given that each of these antibodies can bind to human LAG-3, the V_(H)and V_(L) sequences can be “mixed and matched” to create otheranti-LAG-3 binding molecules of the invention. Preferably, when V_(H)and V_(L) chains are mixed and matched, a V_(H) sequence from aparticular V_(H)/V_(L) pairing is replaced with a structurally similarV_(H) sequence. Likewise, preferably a V_(L) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(L)sequence.

Accordingly, in one aspect, this disclosure provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

(a) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 37-42; and

(b) a light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 43-48;

wherein the antibody specifically binds human LAG-3.

Preferred heavy and light chain combinations include:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 37 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 43;

(b) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 38 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 44;

(c) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 39 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 45;

(d) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 40 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 46;

(e) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 41 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 47; or

(f) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 42 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 48.

In another aspect, this disclosure provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of 25F7, 26H10, 25E3,8B7, 11F2 or 17E5, or combinations thereof. The amino acid sequences ofthe V_(H) CDR1s of 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5 are shown inSEQ ID NOs: 37-42, respectively. The amino acid sequences of the V_(H)CDR2s of 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5 are shown in SEQ ID NOs:43-48, respectively. The amino acid sequences of the V_(H) CDR3s of25F7, 26H10, 25E3, 8B7, 11F2 and 17E5 are shown in SEQ ID NOs: 13-14,GGY and 16-18, respectively. The amino acid sequences of the V_(K) CDR1sof 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5 are shown in SEQ ID NOs: 19-24respectively. The amino acid sequences of the V_(K) CDR2s of 25F7,26H10, 25E3, 8B7, 11F2 and 17E5 are shown in SEQ ID NOs: 25-30. Theamino acid sequences of the V_(K) CDR3s of 25F7, 26H10, 25E3, 8B7, 11F2and 17E5 are shown in SEQ ID NOs: 31-36, respectively. The CDR regionsare delineated using the Kabat system (Kabat et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242).

Given that each of these antibodies can bind to human LAG-3 and thatantigen-binding specificity is provided primarily by the CDR1, CDR2, andCDR3 regions, the V_(H) CDR1, CDR2, and CDR3 sequences and V_(L) CDR1,CDR2, and CDR3 sequences can be “mixed and matched” (i.e., CDRs fromdifferent antibodies can be mixed and match, although each antibody mustcontain a V_(H) CDR1, CDR2, and CDR3 and a V_(L) CDR1, CDR2, and CDR3)to create other anti-LAG-3 binding molecules of the invention. LAG-3binding of such “mixed and matched” antibodies can be tested using thebinding assays described above and in the Examples (e.g., ELISAs,Biacore® analysis). Preferably, when V_(H) CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(H)sequence is replaced with a structurally similar CDR sequence(s).Likewise, when V_(L) CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular V_(L) sequence preferably isreplaced with a structurally similar CDR sequence(s). It will be readilyapparent to the ordinarily skilled artisan that novel V_(H) and V_(L)sequences can be created by substituting one or more V_(H) and/or V_(L)CDR region sequences with structurally similar sequences from the CDRsequences disclosed herein for monoclonal antibodies 25F7, 26H10, 25E3,8B7, 11F2 and 17E5.

Accordingly, in another aspect, this disclosure provides an isolatedmonoclonal antibody, or antigen binding portion thereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-6;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 7-12;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13-14, GGY and 16-18;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 19-24;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 25-30; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 31-36;

wherein the antibody specifically binds human LAG-3.

In a preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 1;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 7;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 13;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 19;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 25; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 31.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 2;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 8;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 14;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 20;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 26; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 32.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 3;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 9;

(c) a heavy chain variable region CDR3 comprising GGY;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 21;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 27; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 33.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 4;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 10;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 16;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 22;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 28; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 34.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 5;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 11;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 17;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 23;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 29; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 35.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 6;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 12;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 18;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 24;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 30; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 36.

It is well known in the art that the CDR3 domain, independently from theCDR1 and/or CDR2 domain(s), alone can determine the binding specificityof an antibody for a cognate antigen and that multiple antibodies canpredictably be generated having the same binding specificity based on acommon CDR3 sequence. See, e.g., Klimka et al., British J. of Cancer83(2):252-260 (2000); Beiboer et al., J. Mol. Biol. 296:833-849 (2000);Rader et al., Proc. Natl. Acad. Sci. U.S.A. 95:8910-8915 (1998); Barbaset al., J. Am. Chem. Soc. 116:2161-2162 (1994); Barbas et al., Proc.Natl. Acad. Sci. U.S.A. 92:2529-2533 (1995); Ditzel et al., J. Immunol.157:739-749 (1996); Berezov et al., BIAjournal 8:Scientific Review 8(2001); Igarashi et al., J. Biochem (Tokyo) 117:452-7 (1995); Bourgeoiset al., J. Virol 72:807-10 (1998); Levi et al., Proc. Natl. Acad. Sci.U.S.A. 90:4374-8 (1993); Polymenis and Stoller, J. Immunol.152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000). Seealso, U.S. Pat. Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216;6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. Each of thesereferences is hereby incorporated by reference in its entirety.

Accordingly, the present disclosure provides monoclonal antibodiescomprising one or more heavy and/or light chain CDR3 domains from anantibody derived from a human or non-human animal, wherein themonoclonal antibody is capable of specifically binding to human LAG-3.Within certain aspects, the present disclosure provides monoclonalantibodies comprising one or more heavy and/or light chain CDR3 domainfrom a non-human antibody, such as a mouse or rat antibody, wherein themonoclonal antibody is capable of specifically binding to LAG-3. Withinsome embodiments, such inventive antibodies comprising one or more heavyand/or light chain CDR3 domain from a non-human antibody (a) are capableof competing for binding with; (b) retain the functionalcharacteristics; (c) bind to the same epitope; and/or (d) have a similarbinding affinity as the corresponding parental non-human antibody.

Within other aspects, the present disclosure provides monoclonalantibodies comprising one or more heavy and/or light chain CDR3 domainfrom a human antibody, such as, e.g., a human antibody obtained from anon-human animal, wherein the human antibody is capable of specificallybinding to human LAG-3. Within other aspects, the present disclosureprovides monoclonal antibodies comprising one or more heavy and/or lightchain CDR3 domain from a first human antibody, such as, for example, ahuman antibody obtained from a non-human animal, wherein the first humanantibody is capable of specifically binding to human LAG-3 and whereinthe CDR3 domain from the first human antibody replaces a CDR3 domain ina human antibody that is lacking binding specificity for LAG-3 togenerate a second human antibody that is capable of specifically bindingto human LAG-3. Within some embodiments, such inventive antibodiescomprising one or more heavy and/or light chain CDR3 domain from thefirst human antibody (a) are capable of competing for binding with; (b)retain the functional characteristics; (c) bind to the same epitope;and/or (d) have a similar binding affinity as the corresponding parentalfirst human antibody.

Antibodies Having Particular Germline Sequences

In certain embodiments, an antibody of the invention comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, this disclosure provides anisolated monoclonal antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region that is the product of orderived from a human V_(H) 3-20 gene, a human V_(H) 4-34 gene, a humanV_(H) 3-33 gene or a human V_(H) 1-24 gene, wherein the antibodyspecifically binds human LAG-3. In another preferred embodiment, thisdisclosure provides an isolated monoclonal antibody, or anantigen-binding portion thereof, comprising a light chain variableregion that is the product of or derived from a human V_(K) L18 gene, ahuman V_(K) L6 gene or a human V_(K) A27 gene, wherein the antibodyspecifically binds human LAG-3. In yet another preferred embodiment,this disclosure provides an isolated monoclonal antibody, orantigen-binding portion thereof, wherein the antibody comprises a heavychain variable region that is the product of or derived from a humanV_(H) 3-20 gene and comprises a light chain variable region that is theproduct of or derived from a human V_(K) L18 gene, wherein the antibodyspecifically binds human LAG-3. In yet another preferred embodiment,this disclosure provides an isolated monoclonal antibody, orantigen-binding portion thereof, wherein the antibody comprises a heavychain variable region that is the product of or derived from a humanV_(H) 4-34 gene and comprises a light chain variable region that is theproduct of or derived from a human V_(K) L6 gene, wherein the antibodyspecifically binds human LAG-3. In yet another preferred embodiment,this disclosure provides an isolated monoclonal antibody, orantigen-binding portion thereof, wherein the antibody comprises a heavychain variable region that is the product of or derived from a humanV_(H) 3-33 gene and comprises a light chain variable region that is theproduct of or derived from a human V_(K) A27 gene, wherein the antibodyspecifically binds human LAG-3. In yet another preferred embodiment,this disclosure provides an isolated monoclonal antibody, orantigen-binding portion thereof, wherein the antibody comprises a heavychain variable region that is the product of or derived from a humanV_(H) 1-24 gene and comprises a light chain variable region that is theproduct of or derived from a human V_(K) L6 gene, wherein the antibodyspecifically binds human LAG-3. In yet another preferred embodiment,this disclosure provides an isolated monoclonal antibody, orantigen-binding portion thereof, wherein the antibody comprises a heavychain variable region that is the product of or derived from a humanV_(H) 3-33 gene and comprises a light chain variable region that is theproduct of or derived from a human V_(K) L6 gene, wherein the antibodyspecifically binds human LAG-3.

Such antibodies can also possess one or more of the functionalcharacteristics described in detail above, such as high affinity bindingto human LAG-3, binding to monkey LAG-3, lack of binding to mouse LAG-3,the ability to inhibit binding of LAG-3 to MHC Class II molecules and/orthe ability to stimulate antigen-specific T cell responses.

An example of an antibody having V_(H) and V_(L) of V_(H) 3-20 and V_(K)L18, respectively, is the 25E3 antibody. Examples of antibodies havingV_(H) and V_(L) of V_(H) 4-34 and V_(K) L6, respectively, are the 25F7and 8B7 antibodies. An example of an antibody having V_(H) and V_(L) ofV_(H) 3-33 and V_(K) A27, respectively, is the 26H10 antibody. Anexample of an antibody having V_(H) and V_(L) of V_(H) 1-24 and V_(K)L6, respectively, is the 11F2 antibody. An example of an antibody havingV_(H) and V_(L) of V_(H) 3-33 and V_(K) L6, respectively, is the 17E5antibody.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e., greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence can contain amino acid differences as compared to the germlinesequence, due to, for example, naturally-occurring somatic mutations orintentional introduction of site-directed mutation. However, a selectedhuman antibody typically is at least 90% identical in amino acidssequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody can be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody candisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of the invention comprises heavyand light chain variable regions comprising amino acid sequences thatare homologous to the amino acid sequences of the preferred antibodiesdescribed herein, and wherein the antibodies retain the desiredfunctional properties of the anti-LAG-3 antibodies of the invention. Forexample, this disclosure provides an isolated monoclonal antibody, orantigen binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 37-42;

(b) the light chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 43-48; and

(c) the antibody specifically binds to human LAG-3.

Additionally or alternatively, the antibody can possess one or more ofthe following functional properties discussed above, such as highaffinity binding to human LAG-3, binding to monkey LAG-3, lack ofbinding to mouse LAG-3, the ability to inhibit binding of LAG-3 to MHCClass II molecules and/or the ability to stimulate antigen-specific Tcell responses.

In various embodiments, the antibody can be, for example, a humanantibody, a humanized antibody or a chimeric antibody.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences can be85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences setforth above. An antibody having V_(H) and V_(L) regions having high(i.e., 80% or greater) homology to the V_(H) and V_(L) regions of thesequences set forth above, can be obtained by mutagenesis (e.g.,site-directed or PCR-mediated mutagenesis) of nucleic acid moleculesencoding SEQ ID NOs: 49-54 or 55-60, followed by testing of the encodedaltered antibody for retained function (i.e., the functions set forthabove) using the functional assays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentdisclosure can further be used as a “query sequence” to perform a searchagainst public databases to, e.g., to identify related sequences. Suchsearches can be performed using the XBLAST program (version 2.0) ofAltschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are useful. See www.ncbi.nlm.nih.gov.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein (e.g.,25F7, 26H10, 25E3, 8B7, 11F2, 17E5), or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the anti-LAG-3 antibodies of the invention. It isunderstood in the art that certain conservative sequence modificationcan be made which do not remove antigen binding. See, e.g., Brummell etal. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng.10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hallet al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993)Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6and Beers et al. (2000) Clin. Can. Res. 6:2835-43. Accordingly, thisdisclosure provides an isolated monoclonal antibody, or antigen bindingportion thereof, comprising a heavy chain variable region comprisingCDR1, CDR2, and CDR3 sequences and a light chain variable regioncomprising CDR1, CDR2, and CDR3 sequences, wherein:

(a) the heavy chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs: 13-14, GGY and 16-18, and conservative modificationsthereof,

(b) the light chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequenceof SEQ ID NOs: 31-36, and conservative modifications thereof, and

(c) the antibody specifically binds human LAG-3.

Additionally or alternatively, the antibody can possess one or more ofthe following functional properties described above, such as highaffinity binding to human LAG-3, binding to monkey LAG-3, lack ofbinding to mouse LAG-3, the ability to inhibit binding of LAG-3 to MHCClass II molecules and/or the ability to stimulate antigen-specific Tcell responses.

In a preferred embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 7-12, and conservative modificationsthereof; and the light chain variable region CDR2 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 25-30, and conservative modifications thereof.In another preferred embodiment, the heavy chain variable region CDR1sequence comprises an amino acid sequence selected from the groupconsisting of amino acid sequences of SEQ ID NOs: 1-6, and conservativemodifications thereof, and the light chain variable region CDR1 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 19-24, and conservativemodifications thereof.

In various embodiments, the antibody can be, for example, humanantibodies, humanized antibodies or chimeric antibodies.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for retainedfunction (i.e., the functions set forth above) using the functionalassays described herein.

Antibodies that Bind to the Same Epitope as Anti-LAG-3 Antibodies

In another embodiment, this disclosure provides antibodies that bind tothe same epitope on LAG-3 as any of the anti-LAG-3 monoclonal antibodiesof the invention (i.e., antibodies that have the ability tocross-compete for binding to human LAG-3 with any of the monoclonalantibodies of the invention). In preferred embodiments, the referenceantibody for cross-competition studies can be the monoclonal antibodies25F7, 26H10, 25E3, 8B7, 11F2 or 17E5.

Such cross-competing antibodies can be identified based on their abilityto cross-compete with 25F7, 26H10, 25E3, 8B7, 11F2 and/or 17E5 instandard LAG-3 binding assays. For example, standard ELISA assays can beused in which a recombinant human LAG-3 protein is immobilized on theplate, one of the antibodies is fluorescently labeled and the ability ofnon-labeled antibodies to compete off the binding of the labeledantibody is evaluated. Additionally or alternatively, BIAcore analysiscan be used to assess the ability of the antibodies to cross-compete.The ability of a test antibody to inhibit the binding of, for example,25F7, 26H10, 25E3, 8B7, 11F2 and/or 17E5, to human LAG-3 demonstratesthat the test antibody can compete with 25F7, 26H10, 25E3, 8B7, 11F2and/or 17E5 for binding to human LAG-3 and thus binds to the sameepitope on human LAG-3 as 25F7, 26H10, 25E3, 8B7, 11F2 and/or 17E5. In apreferred embodiment, the antibody that binds to the same epitope onhuman LAG-3 as 25E3, 25F7, 8B7, 26H10, 11F2 or 17E5 is a humanmonoclonal antibody. Such human monoclonal antibodies can be preparedand isolated as described in the Examples.

As discussed further in Example 3C, the binding of 25E3, 25F7 and 8B7 tohuman LAG-3 has been mapped to an “extra loop” region within the firstextracellular domain of human LAG-3. The sequence of the extra loopregion is set forth in SEQ ID NO: 79. Using a peptide scan experiment,the binding of 25E3 to the extra loop region has been mapped to thefollowing amino acid sequence: PGHPLAPG (SEQ ID NO: 76), whereas thebinding of 25F7 to the extra loop region has been mapped to thefollowing amino acid sequence: HPAAPSSW (SEQ ID NO: 77) and the bindingof 8B7 to the extra loop region has been mapped to the following aminoacid sequence: PAAPSSWG (SEQ ID NO: 78). Accordingly, in a preferredembodiment, the invention provides an anti-LAG-3 antibody that binds anepitope of human LAG-3 comprising the amino acid sequence PGHPLAPG (SEQID NO: 76). In another preferred embodiment, the invention provides ananti-LAG-3 antibody that binds an epitope of human LAG-3 comprising theamino acid sequence HPAAPSSW (SEQ ID NO: 77) or PAAPSSWG (SEQ ID NO:78).

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

In certain embodiments, CDR grafting can be used to engineer variableregions of antibodies. Antibodies interact with target antigenspredominantly through amino acid residues that are located in the sixheavy and light chain complementarity determining regions (CDRs). Forthis reason, the amino acid sequences within CDRs are more diversebetween individual antibodies than sequences outside of CDRs. BecauseCDR sequences are responsible for most antibody-antigen interactions, itis possible to express recombinant antibodies that mimic the propertiesof specific naturally occurring antibodies by constructing expressionvectors that include CDR sequences from the specific naturally occurringantibody grafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann et al. (1998) Nature332:323-327; Jones et al. (1986) Nature 321:522-525; Queen et al. (1989)Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. Nos. 5,225,539;5,530,101; 5,585,089; 5,693,762 and 6,180,370.)

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencescomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-6, SEQ ID NOs: 7-12, and SEQ ID NOs: 13-14, GGY and 16-18,respectively, and a light chain variable region comprising CDR1, CDR2,and CDR3 sequences comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 19-24, SEQ ID NOs: 25-30, and SEQ IDNOs: 31-36, respectively. Thus, such antibodies contain the V_(H) andV_(L) CDR sequences of monoclonal antibodies 25F7, 26H10, 25E3, 8B7,11F2 or 17E5 can contain different framework sequences from theseantibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat et al. (1991), cited supra; Tomlinson et al. (1992)“The Repertoire of Human Germline V_(H) Sequences Reveals about FiftyGroups of V_(H) Segments with Different Hypervariable Loops” J. Mol.Biol. 227:776-798; and Cox et al. (1994) “A Directory of Human Germ-lineV_(H) Segments Reveals a Strong Bias in their Usage” Eur. J. Immunol.24:827-836; the contents of each of which are expressly incorporatedherein by reference. As another example, the germline DNA sequences forhuman heavy and light chain variable region genes can be found in theGenbank database. For example, the following heavy chain germlinesequences found in the HCo7 HuMAb mouse are available in theaccompanying Genbank Accession Nos.: 1-69 (NG_0010109, NT_024637 &BC070333), 3-33 (NG_0010109 & NT_024637) and 3-7 (NG_0010109 &NT_024637). As another example, the following heavy chain germlinesequences found in the HCo12 HuMAb mouse are available in theaccompanying Genbank Accession Nos.: 1-69 (NG_0010109, NT_024637 &BC070333), 5-51 (NG_0010109 & NT_024637), 4-34 (NG_0010109 & NT_024637),3-30.3 (CAJ556644) & 3-23 (AJ406678).

Antibody protein sequences are compared against a compiled proteinsequence database using one of the sequence similarity searching methodscalled the Gapped BLAST (Altschul et al. (1997), supra), which is wellknown to those skilled in the art.

Preferred framework sequences for use in the antibodies of the inventionare those that are structurally similar to the framework sequences usedby selected antibodies of the invention, e.g., similar to the V_(H) 3-20(SEQ ID NO: 69), V_(H) 4-34 (SEQ ID NO: 61), V_(H) 3-33 (SEQ ID NO: 65)or V_(H) 1-24 (SEQ ID NO: 73) framework sequences and/or the V_(K) L18(SEQ ID NO: 71), V_(K) L6 (SEQ ID NO: 63) or V_(K) A27 (SEQ ID NO: 67)framework sequences used by preferred monoclonal antibodies of theinvention. The V_(H) CDR1, CDR2, and CDR3 sequences, and the V_(K) CDR1,CDR2, and CDR3 sequences, can be grafted onto framework regions thathave the identical sequence as that found in the germline immunoglobulingene from which the framework sequence derive, or the CDR sequences canbe grafted onto framework regions that contain one or more mutations ascompared to the germline sequences. For example, it has been found thatin certain instances it is beneficial to mutate residues within theframework regions to maintain or enhance the antigen binding ability ofthe antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedabove) are introduced. The mutations can be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, in another embodiment, the instant disclosure providesisolated anti-LAG-3 monoclonal antibodies, or antigen binding portionsthereof, comprising a heavy chain variable region comprising: (a) aV_(H) CDR1 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-6, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 1-6; (b) a V_(H) CDR2 regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 7-12, or an amino acid sequence having one, two, three, fouror five amino acid substitutions, deletions or additions as compared toSEQ ID NOs: 7-12; (c) a V_(H) CDR3 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 13-14, GGYand 16-18, or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to SEQID NOs: 13-14, GGY and 16-18; (d) a V_(L) CDR1 region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:19-24, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 19-24; (e) a V_(L) CDR2 region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 25-30, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 25-30;and (f) a V_(L) CDR3 region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 31-36, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 31-36.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation cancontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived.

For example, Table A shows regions where a framework region amino acidposition (using Kabat numbering system) differs from the germline andhow this position can be backmutated to the germline by the indicatedsubstitutions:

TABLE A Exemplary Backmutations Framework Amino Acid Position Region(Kabat Numbering) Backmutation 25E3 V_(H) 72 G72R 25E3 V_(H) 95 Y95H25E3 V_(H) 97 T97A 25E3 V_(H) 98 T98R 25F7 V_(H) 69 L69I 25F7 V_(H) 71L71V 25F7 V_(H) 83 R83S 25F7 V_(H) 97 F97R 8B7 V_(H) 76 K76N 8B7 V_(H)79 A79S 8B7 V_(H) 83 N83S 8B7 V_(H) 112 P112Q 11F2 V_(H) 3 D3A 17E5V_(H) 3 H3Q 8B7 V_(H) 59 C59Y 8B7 V_(H) 59 C59S

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention can be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention can bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In a preferred embodiment, the antibody is an IgG4 isotype antibodycomprising a Serine to Proline mutation at a position corresponding toposition 228 (S228P; EU index) in the hinge region of the heavy chainconstant region. This mutation has been reported to abolish theheterogeneity of inter-heavy chain disulfide bridges in the hinge region(Angal et al. supra; position 241 is based on the Kabat numberingsystem). For example, in various embodiments, an anti-LAG-3 antibody ofthe invention can comprise the heavy chain variable region of 25F7 (SEQID NO: 37) or 26H10 (SEQ ID NO: 38) linked to a human IgG4 constantregion in which the Serine at a position corresponding to position 241as described in Angal et al., supra, has been mutated to Proline. Thus,for the 25F7 and 26H10 heavy chain variable regions linked to a humanIgG4 constant region, this mutation corresponds to an S228P mutation bythe EU index.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425. The number of cysteine residues in the hinge region ofCH1 is altered to, for example, facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375. Alternatively, toincrease the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072. Moreover,the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutationsat positions 256, 290, 298, 333, 334 and 339 were shown to improvebinding to FcγRIII. Additionally, the following combination mutants wereshown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224Aand S298A/E333A/K334A.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. See, e.g., U.S. Pat.Nos. 5,714,350 and 6,350,861.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, the cell lines Ms704, Ms705,and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)-fucosyltransferase), such that antibodies expressed in the Ms704,Ms705, and Ms709 cell lines lack fucose on their carbohydrates. TheMs704, Ms705, and Ms709 FUT8^(−/−) cell lines were created by thetargeted disruption of the FUT8 gene in CHO/DG44 cells using tworeplacement vectors (see U.S. Patent Publication No. 20040110704 andYamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As anotherexample, EP 1,176,195 describes a cell line with a functionallydisrupted FUT8 gene, which encodes a fucosyl transferase, such thatantibodies expressed in such a cell line exhibit hypofucosylation byreducing or eliminating the α-1,6 bond-related enzyme. EP 1,176,195 alsodescribes cell lines which have a low enzyme activity for adding fucoseto the N-acetylglucosamine that binds to the Fc region of the antibodyor does not have the enzyme activity, for example the rat myeloma cellline YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 describes avariant CHO cell line, Lec13 cells, with reduced ability to attachfucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields et al. (2002) J. Biol. Chem. 277:26733-26740). Antibodies with amodified glycosylation profile can also be produced in chicken eggs, asdescribed in PCT Publication WO 06/089231. Alternatively, antibodieswith a modified glycosylation profile can be produced in plant cells,such as Lemna. Methods for production of antibodies in a plant systemare disclosed in the U.S. Patent application corresponding to Alston &Bird LLP attorney docket No. 040989/314911, filed on Aug. 11, 2006. PCTPublication WO 99/54342 describes cell lines engineered to expressglycoprotein-modifying glycosyl transferases (e.g.,β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).Alternatively, the fucose residues of the antibody can be cleaved offusing a fucosidase enzyme; e.g., the fucosidase α-L-fucosidase removesfucosyl residues from antibodies (Tarentino et al. (1975) Biochem.14:5516-23).

Another modification of the antibodies herein that is contemplated bythis disclosure is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See, e.g., EP 0 154 316 and EP 0 401384.

Antibody Physical Properties

Antibodies of this disclosure can be characterized by their variousphysical properties, to detect and/or differentiate different classesthereof.

Antibodies of the present disclosure can contain one or moreglycosylation sites in either the light or heavy chain variable region.Such glycosylation sites may result in increased immunogenicity of theantibody or an alteration of the pK of the antibody due to alteredantigen binding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Galaand Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J ExpMed 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al(1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. In some instances, it is preferred to have an anti-LAG-3antibody that does not contain variable region glycosylation. This canbe achieved either by selecting antibodies that do not contain theglycosylation motif in the variable region or by mutating residueswithin the glycosylation region.

In a preferred embodiment, the antibodies of the present disclosure donot contain asparagine isomerism sites. The deamidation of asparaginemay occur on N-G or D-G sequences and result in the creation of anisoaspartic acid residue that introduces a kink into the polypeptidechain and decreases its stability (isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pI), which generallyfalls in the pH range between 6 and 9.5. The pI for an IgG1 antibodytypically falls within the pH range of 7-9.5 and the pI for an IgG4antibody typically falls within the pH range of 6-8. There isspeculation that antibodies with a pI outside the normal range may havesome unfolding and instability under in vivo conditions. Thus, it ispreferred to have an anti-LAG-3 antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range or by mutating charged surfaceresidues.

Each antibody will have a characteristic melting temperature, with ahigher melting temperature indicating greater overall stability in vivo(Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71).Generally, it is preferred that the T_(M1) (the temperature of initialunfolding) be greater than 60° C., preferably greater than 65° C., evenmore preferably greater than 70° C. The melting point of an antibody canbe measured using differential scanning calorimetry (Chen et al (2003)Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) orcircular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

In a preferred embodiment, antibodies are selected that do not degraderapidly. Degradation of an antibody can be measured using capillaryelectrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995)Anal Chem 67:3626-32).

In another preferred embodiment, antibodies are selected that haveminimal aggregation effects, which can lead to the triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation can be measured by several techniques, includingsize-exclusion column (SEC), high performance liquid chromatography(HPLC), and light scattering.

Methods of Engineering Antibodies

As discussed above, the anti-LAG-3 antibodies having V_(H) and V_(L)sequences disclosed herein can be used to create new anti-LAG-3antibodies by modifying the V_(H) and/or V_(L) sequences, or theconstant region(s) attached thereto. Thus, in another aspect of theinvention, the structural features of an anti-LAG-3 antibody of theinvention, e.g. 25F7, 26H10, 25E3, 8B7, 11F2 or 17E5, are used to createstructurally related anti-LAG-3 antibodies that retain at least onefunctional property of the antibodies of the invention, such as bindingto human LAG-3. For example, one or more CDR regions of 25F7, 26H10,25E3, 8B7, 11F2 or 17E5, or mutations thereof, can be combinedrecombinantly with known framework regions and/or other CDRs to createadditional, recombinantly-engineered, anti-LAG-3 antibodies of theinvention, as discussed above. Other types of modifications includethose described in the previous section. The starting material for theengineering method is one or more of the V_(H) and/or V_(L) sequencesprovided herein, or one or more CDR regions thereof. To create theengineered antibody, it is not necessary to actually prepare (i.e.,express as a protein) an antibody having one or more of the V_(H) and/orV_(L) sequences provided herein, or one or more CDR regions thereof.Rather, the information contained in the sequence(s) is used as thestarting material to create a “second generation” sequence(s) derivedfrom the original sequence(s) and then the “second generation”sequence(s) is prepared and expressed as a protein.

Accordingly, in another embodiment, this disclosure provides a methodfor preparing an anti-LAG-3 antibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 sequence selected from the group consisting of SEQ IDNOs: 1-6, a CDR2 sequence selected from the group consisting of SEQ IDNOs: 7-12, and/or a CDR3 sequence selected from the group consisting ofSEQ ID NOs: 13-14, GGY and 16-18; and/or (ii) a light chain variableregion antibody sequence comprising a CDR1 sequence selected from thegroup consisting of SEQ ID NOs: 19-24, a CDR2 sequence selected from thegroup consisting of SEQ ID NOs: 25-30, and/or a CDR3 sequence selectedfrom the group consisting of SEQ ID NOs: 31-36;

(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-LAG-3 antibodies described herein, which functional propertiesinclude, but are not limited to:

(i) high affinity binding to human LAG-3;

(ii) binding to monkey LAG-3;

(iii) lack of binding to mouse LAG-3

(iv) an ability to inhibit binding of LAG-3 to MHC Class II molecules;and/or

(v) an ability to stimulate an immune response (e.g., anantigen-specific T cell response).

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples.

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-LAG-3 antibody coding sequence and the resultingmodified anti-LAG-3 antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art (see, e.g., PCT Publications WO02/092780 and WO 03/074679).

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies of the invention. The nucleic acids can be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, Ausubel, et al., ed. (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York. Anucleic acid of the invention can be, e.g., DNA or RNA and may or maynot contain intronic sequences. In a preferred embodiment, the nucleicacid is a cDNA molecule.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), a nucleic acid encoding such antibodies can be recoveredfrom the gene library.

Preferred nucleic acids molecules of the invention are those encodingthe V_(H) and V_(L) sequences of the 25E3, 25F7, 8B7, 26H10, 11F2 and17E5 monoclonal antibodies. DNA sequences encoding the V_(H) sequencesof 25E3, 25F7, 8B7, 26H10, 11F2 and 17E5 are shown in SEQ ID NOs: 49-54,respectively. DNA sequences encoding the V_(L) sequences of 25E3, 25F7,8B7, 26H10, 11F2 and 17E5 are shown in SEQ ID NOs: 55-60, respectively.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat et al. (1991), supra) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the V_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabatet al., supra) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. In preferred embodiments, thelight chain constant region can be a kappa or lambda constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature348:552-554).

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) of the present disclosure can be producedusing the well-known somatic cell hybridization (hybridoma) technique ofKohler and Milstein (1975) Nature 256: 495. Other embodiments forproducing monoclonal antibodies include viral or oncogenictransformation of B lymphocytes and phage display techniques. Chimericor humanized antibodies are also well known in the art. See e.g., U.S.Pat. Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and6,180,370, the contents of which are specifically incorporated herein byreference in their entirety.

In a preferred embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againsthuman LAG-3 can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as the HuMAb Mouse® and KM Mouse®, respectively, and arecollectively referred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal antibodies (Lonberg et al. (1994), supra; reviewed in Lonberg(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding andLonberg (1995) Ann. N.Y. Acad. Sci. 764:536-546). Preparation and use ofthe HuMAb Mouse®, and the genomic modifications carried by such mice, isfurther described in Taylor et al. (1992) Nucleic Acids Research20:6287-6295; Chen et al. (1993) International Immunology 5: 647-656;Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi etal. (1993) Nature Genetics 4:117-123; Chen et al. (1993) EMBO J. 12:821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor et al.(1994) International Immunology 6: 579-591; and Fishwild et al. (1996)Nature Biotechnology 14: 845-851, the contents of all of which arehereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and5,545,807; PCT Publication Nos. WO 92/03918; WO 93/12227; WO 94/25585;WO 97/13852; WO 98/24884; WO 99/45962 and WO 01/14424, the contents ofwhich are incorporated herein by reference in their entirety.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. This mouse isreferred to herein as a “KM Mouse®,” and is described in detail in PCTPublication WO 02/43478. A modified form of this mouse, which furthercomprises a homozygous disruption of the endogenous FcγRIIB receptorgene, is also described in PCT Publication WO 02/43478 and referred toherein as a “KM/FCGR2D Mouse®.” In addition, mice with either the HCo7or HCo12 heavy chain transgenes or both can be used.

Additional transgenic animal embodiments include the Xenomouse (Abgenix,Inc., U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and6,162,963). Further embodiments include “TC mice” (Tomizuka et al.(2000) Proc. Natl. Acad. Sci. USA 97:722-727) and cows carrying humanheavy and light chain transchromosomes (Kuroiwa et al. (2002) NatureBiotechnology 20:889-894; PCT Publication WO 02/092812). The contents ofthese patents and publications are specifically incorporated herein byreference in their entirety.

In one embodiment, human monoclonal antibodies of the invention areprepared using phage display methods for screening libraries of humanimmunoglobulin genes. See, e.g. U.S. Pat. Nos. 5,223,409; 5,403,484;5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197; 5,885,793;6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081, the contentsof which are incorporated herein by reference in their entirety.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. See,e.g., U.S. Pat. Nos. 5,476,996 and 5,698,767, the contents of which areincorporated herein by reference in their entirety.

In another embodiment, human anti-LAG-3 antibodies are prepared usingphage display where the phages comprise nucleic acids encodingantibodies generated in transgenic animals previously immunized withLAG-3. In a preferred embodiment, the transgenic animal is a HuMab, KM,or Kirin mouse. See, e.g. U.S. Pat. No. 6,794,132, the contents of whichare incorporated herein by reference in its entirety.

Immunization of Human Ig Mice

In one embodiment of the invention, human Ig mice are immunized with apurified or enriched preparation of a LAG-3 antigen, recombinant LAG-3protein, or cells expressing a LAG-3 protein. See, e.g., Lonberg et al.(1994), supra; Fishwild et al. (1996), supra; PCT Publications WO98/24884 or WO 01/14424, the contents of which are incorporated hereinby reference in their entirety. In a preferred embodiment, 6-16 week oldmice are immunized with 5-50 μg of LAG-3 protein. Alternatively, aportion of LAG-3 fused to a non-LAG-3 polypeptide is used.

In one embodiment, the transgenic mice are immunized intraperitoneally(IP) or intravenously (IV) with LAG-3 antigen in complete Freund'sadjuvant, followed by subsequent IP or IV immunizations with antigen inincomplete Freund's adjuvant. In other embodiments, adjuvants other thanFreund's or whole cells in the absence of adjuvant are used. The plasmacan be screened by ELISA and cells from mice with sufficient titers ofanti-LAG-3 human immunoglobulin can be used for fusions.

Generation of Hybridomas Producing Human Monoclonal Antibodies of theInvention

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. Generation of hybridomasis well-known in the art. See, e.g., Harlow and Lane (1988) Antibodies,A Laboratory Manual, Cold Spring Harbor Publications, New York.

Generation of Transfectomas Producing Monoclonal Antibodies of theInvention

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNAencoding partial or full-length light and heavy chains obtained bystandard molecular biology techniques is inserted into one or moreexpression vectors such that the genes are operatively linked totranscriptional and translational regulatory sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene.

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals) that control the transcription or translation of the antibodychain genes. Such regulatory sequences are described, e.g., in Goeddel(Gene Expression Technology. Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990)). Preferred regulatory sequences for mammalianhost cell expression include viral elements that direct high levels ofprotein expression in mammalian cells, such as promoters and/orenhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40),adenovirus, (e.g., the adenovirus major late promoter (AdMLP) andpolyoma. Alternatively, nonviral regulatory sequences can be used, suchas the ubiquitin promoter or β-globin promoter. Still further,regulatory elements composed of sequences from different sources, suchas the SRα promoter system, which contains sequences from the SV40 earlypromoter and the long terminal repeat of human T cell leukemia virustype 1 (Takebe et al. (1988) Mol. Cell. Biol. 8:466-472). The expressionvector and expression control sequences are chosen to be compatible withthe expression host cell used.

The antibody light chain gene and the antibody heavy chain gene can beinserted into the same or separate expression vectors. In preferredembodiments, the variable regions are used to create full-lengthantibody genes of any antibody isotype by inserting them into expressionvectors already encoding heavy chain constant and light chain constantregions of the desired isotype such that the V_(H) segment isoperatively linked to the C_(H) segment(s) within the vector and theV_(L) segment is operatively linked to the C_(L) segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216; 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr⁻ CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl.Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g.,as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Characterization of Antibody Binding to Antigen

Antibodies of the invention can be tested for binding to human LAG-3 by,for example, standard ELISA. Anti-LAG-3 human IgGs can be further testedfor reactivity with a LAG-3 antigen by Western blotting. The bindingspecificity of an antibody of the invention can also be determined bymonitoring binding of the antibody to cells expressing a LAG-3 protein,e.g., flow cytometry. These methods are known in the art. See, e.g.,Harlow and Lane (1988), cited supra.

Immunoconjugates

Antibodies of this invention can be conjugated to a therapeutic agent toform an immunoconjugate such as an antibody-drug conjugate (ADC).Suitable therapeutic agents include antimetabolites, alkylating agents,DNA minor groove binders, DNA intercalators, DNA crosslinkers, histonedeacetylase inhibitors, nuclear export inhibitors, proteasomeinhibitors, topoisomerase I or II inhibitors, heat shock proteininhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitoticagents. In the ADC, the antibody and therapeutic agent preferably areconjugated via a linker cleavable such as a peptidyl, disulfide, orhydrazone linker. More preferably, the linker is a peptidyl linker suchas Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ IDNO:15), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys,Cit, Ser, or Glu. The ADCs can be prepared as described in U.S. Pat.Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02/096910;WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; and WO08/103693; U.S. Patent Publications 20060024317; 20060004081; and20060247295; the disclosures of which are incorporated herein byreference.

Bispecific Molecules

In another aspect, the present disclosure features bispecific moleculescomprising an anti-LAG-3 antibody linked to at least one otherfunctional molecule, e.g., another peptide or protein (e.g., anotherantibody or ligand for a receptor) to generate a bispecific moleculethat binds to at least two different binding sites or target molecules.Thus, as used herein, “bispecific molecule” includes molecules that havethree or more specificities. In a preferred embodiment, the bispecificmolecule comprises a first binding specificity for LAG-3 and a secondbinding specificity for a triggering molecule that recruits cytotoxiceffector cells that can kill a LAG-3 expressing target cell. Examples ofsuitable triggering molecules are CD64, CD89, CD16, and CD3. See, e.g.,Kufer et al., TRENDS in Biotechnology, 22 (5), 238-244 (2004).

In an embodiment, a bispecific molecule has, in addition to an anti-Fcbinding specificity and an anti-LAG-3 binding specificity, a thirdspecificity. The third specificity can be for an anti-enhancement factor(EF), e.g., a molecule that binds to a surface protein involved incytotoxic activity and thereby increases the immune response against thetarget cell. For example, the anti-enhancement factor can bind acytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, or ICAM-1) orother immune cell, resulting in an increased immune response against thetarget cell.

Bispecific molecules can come in many different formats and sizes. Atone end of the size spectrum, a bispecific molecule retains thetraditional antibody format, except that, instead of having two bindingarms of identical specificity, it has two binding arms each having adifferent specificity. At the other extreme are bispecific moleculesconsisting of two single-chain antibody fragments (scFv's) linked by apeptide chain, a so-called Bs(scFv)₂ construct. Intermediate-sizedbispecific molecules include two different F(ab) fragments linked by apeptidyl linker. Bispecific molecules of these and other formats can beprepared by genetic engineering, somatic hybridization, or chemicalmethods. See, e.g., Kufer et al, cited supra; Cao and Suresh,Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al.,Immunology Today, 21 (8), 391-397 (2000), and the references citedtherein.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising an antibody of the present disclosure formulatedtogether with a pharmaceutically acceptable carrier. It may optionallycontain one or more additional pharmaceutically active ingredients, suchas another antibody or a drug. The pharmaceutical compositions of theinvention also can be administered in a combination therapy with, forexample, another immunostimulatory agent, anti-cancer agent, ananti-viral agent, or a vaccine, such that the anti-LAG-3 antibodyenhances the immune response against the vaccine.

The pharmaceutical composition can comprise any number of excipients.Excipients that can be used include carriers, surface active agents,thickening or emulsifying agents, solid binders, dispersion orsuspension aids, solubilizers, colorants, flavoring agents, coatings,disintegrating agents, lubricants, sweeteners, preservatives, isotonicagents, and combinations thereof. The selection and use of suitableexcipients is taught in Gennaro, ed., Remington: The Science andPractice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), thedisclosure of which is incorporated herein by reference.

Preferably, a pharmaceutical composition is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active compound can be coated in a material toprotect it from the action of acids and other natural conditions thatmay inactivate it. The phrase “parenteral administration” as used hereinmeans modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal injection and infusion.Alternatively, an antibody of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, e.g., intranasally, orally, vaginally, rectally,sublingually or topically.

The pharmaceutical compounds of the invention can be in the form ofpharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects.Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydroiodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

Pharmaceutical compositions can be in the form of sterile aqueoussolutions or dispersions. They can also be formulated in amicroemulsion, liposome, or other ordered structure suitable to highdrug concentration.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated and the particular mode of administration and willgenerally be that amount of the composition which produces a therapeuticeffect. Generally, out of one hundred percent, this amount will rangefrom about 0.01% to about ninety-nine percent of active ingredient,preferably from about 0.1% to about 70%, most preferably from about 1%to about 30% of active ingredient in combination with a pharmaceuticallyacceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus can beadministered, several divided doses can be administered over time or thedose can be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Alternatively,antibody can be administered as a sustained release formulation, inwhich case less frequent administration is required.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-LAG-3antibody of the invention include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks. In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

A “therapeutically effective dosage” of an anti-LAG-3 antibody of theinvention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of tumor-bearing subjects, a“therapeutically effective dosage” preferably inhibits tumor growth byat least about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. A therapeutically effectiveamount of a therapeutic compound can decrease tumor size, or otherwiseameliorate symptoms in a subject, which is typically a human or can beanother mammal.

The pharmaceutical composition can be a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered via medical devices such as(1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos.5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and4,596,556); (2) micro-infusion pumps (U.S. Pat. No. 4,487,603); (3)transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion apparati(U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S.Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which areincorporated herein by reference.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example, toensure that the therapeutic compounds of the invention cross theblood-brain barrier, they can be formulated in liposomes, which mayadditionally comprise targeting moieties to enhance selective transportto specific cells or organs. See, e.g. U.S. Pat. Nos. 4,522,811;5,374,548; 5,416,016; and 5,399,331; V. V. Ranade (1989) J. Clin.Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038; Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al.(1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al. (1995) Am.J. Physiol. 1233:134; Schreier et al. (1994) J. Biol. Chem. 269:9090;Keinanen and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler(1994) Immunomethods 4:273.

Uses and Methods of the Invention

The antibodies, antibody compositions and methods of the presentinvention have numerous in vitro and in vivo utilities involving, forexample, detection of LAG-3 or enhancement of immune response byblockade of LAG-3. In a preferred embodiment, the antibodies of thepresent invention are human antibodies. For example, these molecules canbe administered to cells in culture, in vitro or ex vivo, or to humansubjects, e.g., in vivo, to enhance immunity in a variety of situations.Accordingly, in one aspect, the invention provides a method of modifyingan immune response in a subject comprising administering to the subjectthe antibody, or antigen-binding portion thereof, of the invention suchthat the immune response in the subject is modified. Preferably, theresponse is enhanced, stimulated or up-regulated.

Preferred subjects include human patients in need of enhancement of animmune response. The methods are particularly suitable for treatinghuman patients having a disorder that can be treated by augmenting animmune response (e.g., the T-cell mediated immune response). In aparticular embodiment, the methods are particularly suitable fortreatment of cancer in vivo. To achieve antigen-specific enhancement ofimmunity, the anti-LAG-3 antibodies can be administered together with anantigen of interest or the antigen may already be present in the subjectto be treated (e.g., a tumor-bearing or virus-bearing subject). Whenantibodies to LAG-3 are administered together with another agent, thetwo can be administered in either order or simultaneously.

The invention further provides methods for detecting the presence ofhuman LAG-3 antigen in a sample, or measuring the amount of human LAG-3antigen, comprising contacting the sample, and a control sample, with ahuman monoclonal antibody, or an antigen binding portion thereof, whichspecifically binds to human LAG-3, under conditions that allow forformation of a complex between the antibody or portion thereof and humanLAG-3. The formation of a complex is then detected, wherein a differencecomplex formation between the sample compared to the control sample isindicative the presence of human LAG-3 antigen in the sample. Moreover,the anti-LAG-3 antibodies of the invention can be used to purify humanLAG-3 via immunoaffinity purification.

Given the ability of anti-LAG-3 antibodies of the invention to inhibitthe binding of LAG-3 to MHC Class II molecules and to stimulateantigen-specific T cell responses, the invention also provides in vitroand in vivo methods of using the antibodies of the invention tostimulate, enhance or upregulate antigen-specific T cell responses. Forexample, the invention provides a method of stimulating anantigen-specific T cell response comprising contacting said T cell withthe antibody of the invention such that an antigen-specific T cellresponse is stimulated. Any suitable indicator of an antigen-specific Tcell response can be used to measure the antigen-specific T cellresponse. Non-limiting examples of such suitable indicators includeincreased T cell proliferation in the presence of the antibody and/orincrease cytokine production in the presence of the antibody. In apreferred embodiment, interleukin-2 production by the antigen-specific Tcell is stimulated.

The invention also provides a method of stimulating an immune response(e.g., an antigen-specific T cell response) in a subject comprisingadministering an antibody of the invention to the subject such that animmune response (e.g., an antigen-specific T cell response) in thesubject is stimulated. In a preferred embodiment, the subject is atumor-bearing subject and an immune response against the tumor isstimulated. In another preferred embodiment, the subject is avirus-bearing subject and an immune response against the virus isstimulated.

In another aspect, the invention provides a method for inhibiting growthof tumor cells in a subject comprising administering to the subject anantibody of the invention such that growth of the tumor is inhibited inthe subject. In yet another aspect, the invention provides a method oftreating viral infection in a subject comprising administering to thesubject an antibody of the invention such that the viral infection istreated in the subject.

These and other methods of the invention are discussed in further detailbelow.

Cancer

Blockade of LAG-3 by antibodies can enhance the immune response tocancerous cells in the patient. In one aspect, the present inventionrelates to treatment of a subject in vivo using an anti-LAG-3 antibodysuch that growth of cancerous tumors is inhibited. An anti-LAG-3antibody can be used alone to inhibit the growth of cancerous tumors.Alternatively, an anti-LAG-3 antibody can be used in conjunction withother immunogenic agents, standard cancer treatments, or otherantibodies, as described below.

Accordingly, in one embodiment, the invention provides a method ofinhibiting growth of tumor cells in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-LAG-3antibody, or antigen-binding portion thereof. Preferably, the antibodyis a human anti-LAG-3 antibody (such as any of the human anti-humanLAG-3 antibodies described herein). Additionally or alternatively, theantibody can be a chimeric or humanized anti-LAG-3 antibody.

Preferred cancers whose growth may be inhibited using the antibodies ofthe invention include cancers typically responsive to immunotherapy.Non-limiting examples of preferred cancers for treatment includemelanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clearcell carcinoma), prostate cancer (e.g. hormone refractory prostateadenocarcinoma), breast cancer, colon cancer and lung cancer (e.g.non-small cell lung cancer). Additionally, the invention includesrefractory or recurrent malignancies whose growth may be inhibited usingthe antibodies of the invention.

Examples of other cancers that can be treated using the methods of theinvention include bone cancer, pancreatic cancer, skin cancer, cancer ofthe head or neck, cutaneous or intraocular malignant melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, testicular cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin'slymphoma, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, chronic or acute leukemiasincluding acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors ofchildhood, lymphocytic lymphoma, cancer of the bladder, cancer of thekidney or ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinalaxis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. The present invention is also useful fortreatment of metastatic cancers, especially metastatic cancers thatexpress PD-L1 (Iwai et al. (2005) Int. Immunol. 17:133-144).

Optionally, antibodies to LAG-3 can be combined with an immunogenicagent, such as cancerous cells, purified tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules), cells, andcells transfected with genes encoding immune stimulating cytokines (Heet al (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumorvaccines that can be used include peptides of melanoma antigens, such aspeptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, ortumor cells transfected to express the cytokine GM-CSF (discussedfurther below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. By raising the threshold of T cell activation by LAG-3blockade, the tumor responses in the host can be activated.

LAG-3 blockade is likely to be more effective when combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see Rosenberg, S., 2000, Developmentof Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C.,2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCOEducational Book Spring: 414-428; Foon, K. 2000, ASCO Educational BookSpring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines,Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principlesand Practice of Oncology, Fifth Edition). In one of these strategies, avaccine is prepared using autologous or allogeneic tumor cells. Thesecellular vaccines have been shown to be most effective when the tumorcells are transduced to express GM-CSF. GM-CSF has been shown to be apotent activator of antigen presentation for tumor vaccination (Dranoffet al. (1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. LAG-3 blockade can be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen can include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al. (1994) Science266: 2011-2013). (These somatic tissues may be protected from immuneattack by various means). Tumor antigen can also be “neo-antigens”expressed in cancer cells because of somatic mutations that alterprotein sequence or create fusion proteins between two unrelatedsequences (i.e., bcr-abl in the Philadelphia chromosome), or idiotypefrom B cell tumors.

Other tumor vaccines can include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which can be used in conjunction with LAG-3blockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot & Srivastava(1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withLAG-3 blockade to activate more potent anti-tumor responses.

LAG-3 blockade can also be combined with standard cancer treatments.LAG-3 blockade can be effectively combined with chemotherapeuticregimes. In these instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr et al. (1998) CancerResearch 58: 5301-5304). An example of such a combination is ananti-LAG-3 antibody in combination with decarbazine for the treatment ofmelanoma. Another example of such a combination is an anti-LAG-3antibody in combination with interleukin-2 (IL-2) for the treatment ofmelanoma. The scientific rationale behind the combined use of LAG-3blockade and chemotherapy is that cell death, that is a consequence ofthe cytotoxic action of most chemotherapeutic compounds, should resultin increased levels of tumor antigen in the antigen presentationpathway. Other combination therapies that may result in synergy withLAG-3 blockade through cell death are radiation, surgery, and hormonedeprivation. Each of these protocols creates a source of tumor antigenin the host. Angiogenesis inhibitors can also be combined with LAG-3blockade. Inhibition of angiogenesis leads to tumor cell death which mayfeed tumor antigen into host antigen presentation pathways.

LAG-3 blocking antibodies can also be used in combination withbispecific antibodies that target Fcα or Fcγ receptor-expressingeffectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. For example anti-Fc receptor/anti tumor antigen (e.g.,Her-2/neu) bispecific antibodies have been used to target macrophages tosites of tumor. This targeting may more effectively activate tumorspecific responses. The T cell arm of these responses would be augmentedby the use of LAG-3 blockade. Alternatively, antigen may be delivereddirectly to DCs by the use of bispecific antibodies which bind to tumorantigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-β (Kehrl et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365).Antibodies to each of these entities can be used in combination withanti-LAG-3 to counteract the effects of the immunosuppressive agent andfavor tumor immune responses by the host.

Other antibodies which activate host immune responsiveness can be usedin combination with anti-LAG-3. These include molecules on the surfaceof dendritic cells which activate DC function and antigen presentation.Anti-CD40 antibodies are able to substitute effectively for T cellhelper activity (Ridge et al. (1998) Nature 393: 474-478) and can beused in conjunction with LAG-3 antibodies (Ito et al. (2000)Immunobiology 201 (5) 527-40). Activating antibodies to T cellcostimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097),OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero etal. (1997) Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff et al.(1999) Nature 397: 262-266) may also provide for increased levels of Tcell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. LAG-3 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to stimulateantigen-specific T cells against tumor (Greenberg & Riddell (1999)Science 285: 546-51). These methods can also be used to activate T cellresponses to infectious agents such as CMV. Ex vivo activation in thepresence of anti-LAG-3 antibodies can increase the frequency andactivity of the adoptively transferred T cells.

Infectious Diseases

Other methods of the invention are used to treat patients that have beenexposed to particular toxins or pathogens. Accordingly, another aspectof the invention provides a method of treating an infectious disease ina subject comprising administering to the subject an anti-LAG-3antibody, or antigen-binding portion thereof, such that the subject istreated for the infectious disease. Preferably, the antibody is a humananti-human LAG-3 antibody (such as any of the human anti-LAG-3antibodies described herein). Additionally or alternatively, theantibody can be a chimeric or humanized antibody.

Similar to its application to tumors as discussed above, antibodymediated LAG-3 blockade can be used alone, or as an adjuvant, incombination with vaccines, to stimulate the immune response topathogens, toxins, and self-antigens. Examples of pathogens for whichthis therapeutic approach can be particularly useful, include pathogensfor which there is currently no effective vaccine, or pathogens forwhich conventional vaccines are less than completely effective. Theseinclude, but are not limited to HIV, Hepatitis (A, B, & C), Influenza,Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonasaeruginosa. LAG-3 blockade is particularly useful against establishedinfections by agents such as HIV that present altered antigens over thecourse of the infections. These novel epitopes are recognized as foreignat the time of anti-human LAG-3 administration, thus provoking a strongT cell response that is not dampened by negative signals through LAG-3.

Some examples of pathogenic viruses causing infections treatable bymethods of the invention include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the invention include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the invention include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the invention include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, Nippostrongylus brasiliensis.

In all of the above methods, LAG-3 blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens (see, e.g., Holliger (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure2:1121-1123).

Autoimmune Reactions

Anti-LAG-3 antibodies may provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many anti-tumor responses involve anti-selfreactivities (van Elsas et al. (2001) J. Exp. Med. 194:481-489;Overwijk, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987;Hurwitz, (2000) supra; Rosenberg & White (1996) J. Immunother EmphasisTumor Immunol 19 (1): 81-4). Therefore, it is possible to consider usinganti-LAG-3 blockade in conjunction with various self proteins in orderto devise vaccination protocols to efficiently generate immune responsesagainst these self proteins for disease treatment. For example,Alzheimer's disease involves inappropriate accumulation of Aβ peptide inamyloid deposits in the brain; antibody responses against amyloid areable to clear these amyloid deposits (Schenk et al., (1999) Nature 400:173-177).

Other self proteins can also be used as targets such as IgE for thetreatment of allergy and asthma, and TNFα for rheumatoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of anti-LAG-3 antibody. Neutralizing antibody responses toreproductive hormones can be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors can also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-LAG-3 antibodycan be used for induction of therapeutic autoimmune responses to treatpatients having an inappropriate accumulation of other self-antigens,such as amyloid deposits, including Aβ in Alzheimer's disease, cytokinessuch as TNFα, and IgE.

Vaccines

Anti-LAG-3 antibodies can be used to stimulate antigen-specific immuneresponses by coadministration of an anti-LAG-3 antibody with an antigenof interest (e.g., a vaccine). Accordingly, in another aspect theinvention provides a method of enhancing an immune response to anantigen in a subject, comprising administering to the subject: (i) theantigen; and (ii) an anti-LAG-3 antibody, or antigen-binding portionthereof, such that an immune response to the antigen in the subject isenhanced. Preferably, the antibody is a human anti-human LAG-3 antibody(such as any of the human anti-LAG-3 antibodies described herein).Additionally or alternatively, the antibody can be a chimeric orhumanized antibody. The antigen can be, for example, a tumor antigen, aviral antigen, a bacterial antigen or an antigen from a pathogen.Non-limiting examples of such antigens include those discussed in thesections above, such as the tumor antigens (or tumor vaccines) discussedabove, or antigens from the viruses, bacteria or other pathogensdescribed above.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) of the invention in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, human anti-LAG-3 antibodies of the inventioncan be co-administered with one or other more therapeutic agents, e.g.,a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immuno-complex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, bythemselves, are only effective at levels which are toxic or subtoxic toa patient. Cisplatin is intravenously administered as a 100 mg/ml doseonce every four weeks and adriamycin is intravenously administered as a60-75 mg/ml dose once every 21 days. Co-administration of the humananti-LAG-3 antibodies, or antigen binding fragments thereof, of thepresent invention with chemotherapeutic agents provides two anti-canceragents which operate via different mechanisms which yield a cytotoxiceffect to human tumor cells. Such co-administration can solve problemsdue to development of resistance to drugs or a change in theantigenicity of the tumor cells which would render them unreactive withthe antibody.

Also within the scope of the present invention are kits comprising theantibody compositions of the invention (e.g., human antibodies,bispecific or multispecific molecules, or immunoconjugates) andinstructions for use. The kit can further contain at least oneadditional reagent, or one or more additional human antibodies of theinvention (e.g., a human antibody having a complementary activity whichbinds to an epitope in LAG-3 antigen distinct from the first humanantibody). Kits typically include a label indicating the intended use ofthe contents of the kit. The term label includes any writing, orrecorded material supplied on or with the kit, or which otherwiseaccompanies the kit.

Combination Therapy

In another aspect, the invention provides methods of combination therapyin which an anti-LAG-3 antibody is coadministered with one or moreadditional antibodies that are effective in stimulating immune responsesto thereby further enhance, stimulate or upregulate immune responses ina subject. For example, the invention provides a method for stimulatingan immune response in a subject comprising administering to the subjectan anti-LAG-3 antibody and one or more additional immunostimulatoryantibodies, such as an anti-PD-1 antibody, an anti-PD-L1 antibody and/oran anti-CTLA-4 antibody, such that an immune response is stimulated inthe subject, for example to inhibit tumor growth or to stimulate ananti-viral response. In one embodiment, the subject is administered ananti-LAG-3 antibody and an anti-PD-1 antibody. In another embodiment,the subject is administered an anti-LAG-3 antibody and an anti-PD-L1antibody. In yet another embodiment, the subject is administered ananti-LAG-3 antibody and an anti-CTLA-4 antibody. In one embodiment, theanti-LAG-3 antibody is a human antibody, such as an antibody of thedisclosure. Alternatively, the anti-LAG-3 antibody can be, for example,a chimeric or humanized antibody (e.g., prepared from a mouse anti-LAG-3mAb). In another embodiment, the at least one additionalimmunostimulatory antibody (e.g., anti-PD-1, anti-PD-L1 and/oranti-CTLA-4 antibody) is a human antibody. Alternatively, the at leastone additional immunostimulatory antibody can be, for example, achimeric or humanized antibody (e.g., prepared from a mouse anti-PD-1,anti-PD-L1 and/or anti-CTLA-4 antibody).

In one embodiment, the present invention provides a method for treatinga hyperproliferative disease (e.g., cancer), comprising administering aLAG-3 antibody and a CTLA-4 antibody to a subject. In furtherembodiments, the anti-LAG-3 antibody is administered at a subtherapeuticdose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose,or both are administered at a subtherapeutic dose. In anotherembodiment, the present invention provides a method for altering anadverse event associated with treatment of a hyperproliferative diseasewith an immunostimulatory agent, comprising administering an anti-LAG-3antibody and a subtherapeutic dose of anti-CTLA-4 antibody to a subject.In certain embodiments, the subject is human. In certain embodiments,the anti-CTLA-4 antibody is human sequence monoclonal antibody 10D1(described in PCT Publication WO 01/14424) and the anti-LAG-3 antibodyis human sequence monoclonal antibody, such as 25F7, 26H10, 25E3, 8B7,11F2 or 17E5 described herein. Other anti-CTLA-4 antibodies encompassedby the methods of the present invention include, for example, thosedisclosed in: WO 98/42752; WO 00/37504; U.S. Pat. No. 6,207,156; Hurwitzet al. (1998) Proc. Natl. Acad. Sci. USA 95(17):10067-10071; Camacho etal. (2004) J Clin. Oncology 22(145): Abstract No. 2505 (antibodyCP-675206); and Mokyr et al. (1998) Cancer Res. 58:5301-5304. In certainembodiments, the anti-CTLA-4 antibody binds to human CTLA-4 with a K_(D)of 5×10⁻⁸ M or less, binds to human CTLA-4 with a K_(D) of 1×10⁻⁸ M orless, binds to human CTLA-4 with a K_(D) of 5×10⁻⁹ M or less, or bindsto human CTLA-4 with a K_(D) of between 1×10⁻⁸M and 1×10⁻¹⁰ M or less.

In one embodiment, the present invention provides a method for treatinga hyperproliferative disease (e.g., cancer), comprising administering aLAG-3 antibody and a PD-1 antibody to a subject. In further embodiments,the anti-LAG-3 antibody is administered at a subtherapeutic dose, theanti-PD-1 antibody is administered at a subtherapeutic dose, or both areadministered at a subtherapeutic dose. In another embodiment, thepresent invention provides a method for altering an adverse eventassociated with treatment of a hyperproliferative disease with animmunostimulatory agent, comprising administering an anti-LAG-3 antibodyand a subtherapeutic dose of anti-PD-1 antibody to a subject. In certainembodiments, the subject is human. In certain embodiments, the anti-PD-1antibody is a human sequence monoclonal antibody and the anti-LAG-3antibody is human sequence monoclonal antibody, such as 25F7, 26H10,25E3, 8B7, 11F2 or 17E5 described herein. Examples of human sequenceanti-PD-1 antibodies include 17D8, 2D3, 4H1, 5C4 and 4A11, which aredescribed in PCT Publication WO 06/121168. In certain embodiments, theanti-PD-1 antibody binds to human PD-1 with a K_(D) of 5×10⁻⁸ M or less,binds to human PD-1 with a K_(D) of 1×10⁻⁸ M or less, binds to humanPD-1 with a K_(D) of 5×10⁻⁹ M or less, or binds to human PD-1 with aK_(D) of between 1×10⁻⁸ M and 1×10⁻¹⁰ M or less.

In one embodiment, the present invention provides a method for treatinga hyperproliferative disease (e.g., cancer), comprising administering aLAG-3 antibody and a PD-L1 antibody to a subject. In furtherembodiments, the anti-LAG-3 antibody is administered at a subtherapeuticdose, the anti-PD-L1 antibody is administered at a subtherapeutic dose,or both are administered at a subtherapeutic dose. In anotherembodiment, the present invention provides a method for altering anadverse event associated with treatment of a hyperproliferative diseasewith an immunostimulatory agent, comprising administering an anti-LAG-3antibody and a subtherapeutic dose of anti-PD-L1 antibody to a subject.In certain embodiments, the subject is human. In certain embodiments,the anti-PD-L1 antibody is a human sequence monoclonal antibody and theanti-LAG-3 antibody is human sequence monoclonal antibody, such as 25F7,26H10, 25E3, 8B7, 11F2 or 17E5 described herein. Examples of humansequence anti-PD-L1 antibodies include 3G10, 12A4, 10A5, 5F8, 10H10,1B12, 7H1, 11E6, 12B7 and 13G4, which are described in PCT PublicationWO 07/005874. In certain embodiments, the anti-PD-L1 antibody binds tohuman PD-L1 with a K_(D) of 5×10⁻⁸ M or less, binds to human PD-L1 witha K_(D) of 1×10⁻⁸ M or less, binds to human PD-L1 with a K_(D) of 5×10⁻⁹M or less, or binds to human PD-L1 with a K_(D) of between 1×10⁻⁸ M and1×10⁻¹⁰ M or less.

Blockade of LAG-3 and one or more second target antigens such as CTLA-4and/or PD-1 and/or PD-L1 by antibodies can enhance the immune responseto cancerous cells in the patient. Cancers whose growth may be inhibitedusing the antibodies of the instant disclosure include cancers typicallyresponsive to immunotherapy. Representative examples of cancers fortreatment with the combination therapy of the instant disclosure includethose cancers specifically listed above in the discussion of monotherapywith anti-LAG-3 antibodies.

In certain embodiments, the combination of therapeutic antibodiesdiscussed herein can be administered concurrently as a singlecomposition in a pharmaceutically acceptable carrier, or concurrently asseparate compositions with each antibody in a pharmaceuticallyacceptable carrier. In another embodiment, the combination oftherapeutic antibodies can be administered sequentially. For example, ananti-CTLA-4 antibody and an anti-LAG-3 antibody can be administeredsequentially, such as anti-CTLA-4 antibody being administered first andanti-LAG-3 antibody second, or anti-LAG-3 antibody being administeredfirst and anti-CTLA-4 antibody second. Additionally or alternatively, ananti-PD-1 antibody and an anti-LAG-3 antibody can be administeredsequentially, such as anti-PD-1 antibody being administered first andanti-LAG-3 antibody second, or anti-LAG-3 antibody being administeredfirst and anti-PD-1 antibody second. Additionally or alternatively, ananti-PD-L1 antibody and an anti-LAG-3 antibody can be administeredsequentially, such as anti-PD-L1 antibody being administered first andanti-LAG-3 antibody second, or anti-LAG-3 antibody being administeredfirst and anti-PD-L1 antibody second.

Furthermore, if more than one dose of the combination therapy isadministered sequentially, the order of the sequential administrationcan be reversed or kept in the same order at each time point ofadministration, sequential administrations can be combined withconcurrent administrations, or any combination thereof. For example, thefirst administration of a combination anti-CTLA-4 antibody andanti-LAG-3 antibody can be concurrent, the second administration can besequential with anti-CTLA-4 first and anti-LAG-3 second, and the thirdadministration can be sequential with anti-LAG-3 first and anti-CTLA-4second, etc. Additionally or alternatively, the first administration ofa combination anti-PD-1 antibody and anti-LAG-3 antibody can beconcurrent, the second administration can be sequential with anti-PD-1first and anti-LAG-3 second, and the third administration can besequential with anti-LAG-3 first and anti-PD-1 second, etc. Additionallyor alternatively, the first administration of a combination anti-PD-L1antibody and anti-LAG-3 antibody can be concurrent, the secondadministration can be sequential with anti-PD-L1 first and anti-LAG-3second, and the third administration can be sequential with anti-LAG-3first and anti-PD-L1 second, etc. Another representative dosing schemecan involve a first administration that is sequential with anti-LAG-3first and anti-CTLA-4 (and/or anti-PD-1 and/or anti-PD-L1) second, andsubsequent administrations may be concurrent.

Optionally, the combination of anti-LAG-3 and one or more additionalantibodies (e.g., anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1antibodies) can be further combined with an immunogenic agent, such ascancerous cells, purified tumor antigens (including recombinantproteins, peptides, and carbohydrate molecules), cells, and cellstransfected with genes encoding immune stimulating cytokines (He et al.(2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccinesthat can be used include peptides of melanoma antigens, such as peptidesof gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cellstransfected to express the cytokine GM-CSF (discussed further below). Acombined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade can befurther combined with a vaccination protocol, such as any of thevaccination protocols discussed in detail above with respect tomonotherapy with anti-LAG-3 antibodies.

A combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade can alsobe further combined with standard cancer treatments. For example, acombined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade can beeffectively combined with chemotherapeutic regimes. In these instances,it is possible to reduce the dose of other chemotherapeutic reagentadministered with the combination of the instant disclosure (Mokyr etal. (1998) Cancer Research 58: 5301-5304). An example of such acombination is a combination of anti-LAG-3 and anti-CTLA-4 antibodiesand/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies further incombination with decarbazine for the treatment of melanoma. Anotherexample is a combination of anti-LAG-3 and anti-CTLA-4 antibodies and/oranti-PD-1 antibodies and/or anti-PD-L1 antibodies further in combinationwith interleukin-2 (IL-2) for the treatment of melanoma. The scientificrationale behind the combined use of LAG-3 and CTLA-4 and/or PD-1 and/orPD-L1 blockade with chemotherapy is that cell death, which is aconsequence of the cytotoxic action of most chemotherapeutic compounds,should result in increased levels of tumor antigen in the antigenpresentation pathway. Other combination therapies that may result insynergy with a combined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1blockade through cell death include radiation, surgery, or hormonedeprivation. Each of these protocols creates a source of tumor antigenin the host. Angiogenesis inhibitors can also be combined with acombined LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockade. Inhibitionof angiogenesis leads to tumor cell death, which can be a source oftumor antigen fed into host antigen presentation pathways.

A combination of LAG-3 and CTLA-4 and/or PD-1 and/or PD-L1 blockingantibodies can also be used in combination with bispecific antibodiesthat target Fcα or Fcγ receptor-expressing effector cells to tumor cells(see, e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243). Bispecificantibodies can be used to target two separate antigens. The T cell armof these responses would be augmented by the use of a combined LAG-3 andCTLA-4 and/or PD-1 and/or PD-L1 blockade.

In another example, a combination of anti-LAG-3 and anti-CTLA-4 and/oranti-PD-1 antibodies and/or anti-PD-L1 antibodies can be used inconjunction with anti-neoplastic antibodies, such as Rituxan®(rituximab), Herceptin® (trastuzumab), Bexxar® (tositumomab), Zevalin®(ibritumomab), Campath® (alemtuzumab), Lymphocide® (eprtuzumab),Avastin® (bevacizumab), and Tarceva® (erlotinib), and the like. By wayof example and not wishing to be bound by theory, treatment with ananti-cancer antibody or an anti-cancer antibody conjugated to a toxincan lead to cancer cell death (e.g., tumor cells) which would potentiatean immune response mediated by CTLA-4, PD-1, PD-L1 or LAG-3. In anexemplary embodiment, a treatment of a hyperproliferative disease (e.g.,a cancer tumor) can include an anti-cancer antibody in combination withanti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1antibodies, concurrently or sequentially or any combination thereof,which can potentiate an anti-tumor immune responses by the host.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofproteins, which are expressed by the tumors and which areimmunosuppressive. These include, among others, TGF-β (Kehrl et al.(1986) J Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992)Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996)Science 274: 1363-1365). In another example, antibodies to each of theseentities can be further combined with an anti-LAG-3 and anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 antibody combination to counteractthe effects of immunosuppressive agents and favor anti-tumor immuneresponses by the host.

Other antibodies that can be used to activate host immune responsivenesscan be further used in combination with an anti-LAG-3 and anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 antibody combination. These includemolecules on the surface of dendritic cells that activate DC functionand antigen presentation. Anti-CD40 antibodies (Ridge et al., supra) canbe used in conjunction with an anti-LAG-3 and anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 combination (Ito et al., supra). Otheractivating antibodies to T cell costimulatory molecules Weinberg et al.,supra, Melero et al. supra, Hutloff et al., supra) may also provide forincreased levels of T cell activation.

As discussed above, bone marrow transplantation is currently being usedto treat a variety of tumors of hematopoietic origin. A combined LAG-3and CTLA-4 and/or PD-1 and/or PD-L1 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

Several experimental treatment protocols involve ex vivo activation andexpansion of antigen specific T cells and adoptive transfer of thesecells into recipients in order to antigen-specific T cells against tumor(Greenberg & Riddell, supra). These methods can also be used to activateT cell responses to infectious agents such as CMV. Ex vivo activation inthe presence of anti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 antibodies can be expected to increase the frequency andactivity of the adoptively transferred T cells.

In certain embodiments, the present invention provides a method foraltering an adverse event associated with treatment of ahyperproliferative disease (e.g., cancer) with an immunostimulatoryagent, comprising administering a anti-LAG-3 antibody and asubtherapeutic dose of anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1antibody to a subject. For example, the methods of the present inventionprovide for a method of reducing the incidence of immunostimulatorytherapeutic antibody-induced colitis or diarrhea by administering anon-absorbable steroid to the patient. Because any patient who willreceive an immunostimulatory therapeutic antibody is at risk fordeveloping colitis or diarrhea induced by such an antibody, this entirepatient population is suitable for therapy according to the methods ofthe present invention. Although steroids have been administered to treatinflammatory bowel disease (IBD) and prevent exacerbations of IBD, theyhave not been used to prevent (decrease the incidence of) IBD inpatients who have not been diagnosed with IBD. The significant sideeffects associated with steroids, even non-absorbable steroids, havediscouraged prophylactic use.

In further embodiments, a combination LAG-3 and CTLA-4 and/or PD-1and/or PD-L1 blockade (i.e., immunostimulatory therapeutic antibodiesanti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 antibodies and/or anti-PD-L1antibodies) can be further combined with the use of any non-absorbablesteroid. As used herein, a “non-absorbable steroid” is a glucocorticoidthat exhibits extensive first pass metabolism such that, followingmetabolism in the liver, the bioavailability of the steroid is low,i.e., less than about 20%. In one embodiment of the invention, thenon-absorbable steroid is budesonide. Budesonide is a locally-actingglucocorticosteroid, which is extensively metabolized, primarily by theliver, following oral administration. ENTOCORT EC® (Astra-Zeneca) is apH- and time-dependent oral formulation of budesonide developed tooptimize drug delivery to the ileum and throughout the colon. ENTOCORTEC® is approved in the U.S. for the treatment of mild to moderateCrohn's disease involving the ileum and/or ascending colon. The usualoral dosage of ENTOCORT EC® for the treatment of Crohn's disease is 6 to9 mg/day. ENTOCORT EC® is released in the intestines before beingabsorbed and retained in the gut mucosa. Once it passes through the gutmucosa target tissue, ENTOCORT EC® is extensively metabolized by thecytochrome P450 system in the liver to metabolites with negligibleglucocorticoid activity. Therefore, the bioavailability is low (about10%). The low bioavailability of budesonide results in an improvedtherapeutic ratio compared to other glucocorticoids with less extensivefirst-pass metabolism. Budesonide results in fewer adverse effects,including less hypothalamic-pituitary suppression, thansystemically-acting corticosteroids. However, chronic administration ofENTOCORT EC® can result in systemic glucocorticoid effects such ashypercorticism and adrenal suppression. See PDR 58^(th) ed. 2004;608-610.

In still further embodiments, a combination LAG-3 and CTLA-4 and/or PD-1and/or PD-L1 blockade (i.e., immunostimulatory therapeutic antibodiesanti-LAG-3 and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1antibodies) in conjunction with a non-absorbable steroid can be furthercombined with a salicylate. Salicylates include 5-ASA agents such as,for example: sulfasalazine (AZULFIDINE®, Pharmacia & UpJohn); olsalazine(DIPENTUM®, Pharmacia & UpJohn); balsalazide (COLAZAL®, SalixPharmaceuticals, Inc.); and mesalamine (ASACOL®, Procter & GamblePharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.;ROWASA®, Solvay).

In accordance with the methods of the present invention, a salicylateadministered in combination with anti-LAG-3 and anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 antibodies and a non-absorbable steroid canincludes any overlapping or sequential administration of the salicylateand the non-absorbable steroid for the purpose of decreasing theincidence of colitis induced by the immunostimulatory antibodies. Thus,for example, methods for reducing the incidence of colitis induced bythe immunostimulatory antibodies according to the present inventionencompass administering a salicylate and a non-absorbable concurrentlyor sequentially (e.g., a salicylate is administered 6 hours after anon-absorbable steroid), or any combination thereof. Further, accordingto the present invention, a salicylate and a non-absorbable steroid canbe administered by the same route (e.g., both are administered orally)or by different routes (e.g., a salicylate is administered orally and anon-absorbable steroid is administered rectally), which may differ fromthe route(s) used to administer the anti-LAG-3 and anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 antibodies.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference. In particular, the disclosures of PCTpublications WO 09/045957, WO 09/073533, WO 09/073546, and WO 09/054863are expressly incorporated herein by reference.

Example 1: Generation of Human Monoclonal Antibodies Against LAG-3

Anti-LAG-3 human monoclonal antibodies were generated using transgenicmice that express human antibody genes, as follows.

Antigens

Recombinant human LAG-3 fusion proteins were used as the immunogen toraise anti-human LAG-3 antibodies. In certain immunizations, a fusionprotein comprising the entire extracellular region (domains 1-4) ofhuman LAG-3 fused to a human immunoglobulin Fc domain (R&D Systems,Catalog #2319-L3) (D1-D4 hFc) or a mouse immunoglobulin Fc domain (D1-D4mFc) was used as the immunogen. For other immunizations, a fusionprotein comprising only the first two extracellular domains of humanLAG-3 fused to a mouse immunoglobulin Fc domain (D1-D2 mFc) was used asthe immunogen. The LAG-3 fusion proteins were prepared using standardrecombinant DNA techniques.

Transgenic Transchromosomic KM Mouse™ and KM/FCGR2D Mouse™ Strains

Fully human monoclonal antibodies to human LAG-3 were prepared usingmice of the transgenic transchromosomic KM Mouse™ and KM/FCGR2D Mouse™strains, which expresses human antibody genes.

In the KM Mouse™ strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al. (1993) EMBO J.12:811-820 and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of PCT Publication WO01/09187. Furthermore, this mouse strain carries a human kappa lightchain transgene, KCo5, as described in Fishwild et al., supra. Thestrain also contains the SC20 transchromosome, which carries the humanIg heavy chain locus, as described in PCT Publication WO 02/43478. TheKM/FCGR2D Mouse™ strain is the same as the KM Mouse™ strain except thatits genome also comprises a homozygous disruption of the endogenousFcγRIIB gene. The KM Mouse™ and KM/FCGR2D Mouse™ strains are alsodescribed in detail in U.S. Application Publication No. 20020199213.

KM Mouse™ and KM/FCGR2D Mouse™ Immunizations:

To generate fully human monoclonal antibodies to LAG-3, mice of the KMMouse™ and KM/FCGR2D Mouse™ strains were immunized with one of the threedifferent recombinant LAG-3 fusion protein described above (D1-D4 hFc,D1-D4 mFc, D1-D2, mFc). General immunization schemes are described inLonberg et al. (1994) supra; Fishwild et al., supra and PCT PublicationWO 98/24884. The mice were 6-16 weeks of age upon the first infusion ofantigen. Mice were immunized intraperitoneally (IP) and/orsubcutaneously (SC). The mice were immunized biweekly four times with 10μg of the recombinant LAG-3 fusion protein, followed by immunizationtwice with 20 μg of the same immunogen in Ribi as an adjuvant. Theimmune response was monitored by retroorbital bleeds. The plasma wasscreened by ELISA (as described below), and mice with sufficient titersof anti-LAG-3 human immunoglobulin were used for fusions. Prior tosacrifice and removal of the spleens, the mice were boostedintravenously and intraperitoneally with 20 μg of antigen followed by asubsequent intravenous boost with 20 μg of antigen.

Selection of KM and KM/FCGR2D Mice Producing Anti-LAG-3 Antibodies

To select mice producing antibodies that bound LAG-3 protein, sera frommice immunized with the D1-D4 hFc fusion protein were tested by amodified ELISA as originally described by Fishwild et al. (1996).Briefly, microtiter plates were coated with purified recombinant LAG-3fusion protein at 1 μg/ml in PBS, 50 μl/wells incubated 4° C. overnight,then blocked with 200 μl/well of 5% BSA in PBS. Dilutions of plasma fromLAG-3-immunized mice were added to each well and incubated for 1-2 hoursat ambient temperature. The plates were washed with PBS/Tween and thenincubated with a goat-anti-human kappa light chain polyclonal antibodyconjugated with Horse Radish Peroxidase (HRP) for 1 hour at roomtemperature. After washing, the plates were developed with ABTSsubstrate and analyzed by spectrophotometer at OD 405.

For mice immunized with the D1-D4 mFc or D1-D2 mFc fusion proteins, serafrom these mice with were tested by indirect ELISA using goat anti-mouseIgG to coat the plates for one hour prior to coating with the antigen toeliminate nonspecific binding to the mouse Fc part. Then the same ELISAsteps as described above were carried out.

Mice that developed the highest titers of anti-LAG-3 antibodies wereused for fusions. Fusions were performed as described below andhybridoma supernatants were tested for anti-LAG-3 activity by ELISA.

Generation of Hybridomas Producing Human Monoclonal Antibodies to LAG-3Proteins

The mouse splenocytes, isolated from the KM or KM/FCGR2D mice, werefused by electric field based electrofusion using a Cyto Pulse largechamber cull fusion electroporator (Cyto Pulse Sciences, Inc., GlenBurnie, MD) to a mouse myeloma cell line. The resulting hybridomas werethen screened for the production of antigen-specific antibodies. Singlecell suspensions of splenic lymphocytes from immunized mice were fusedto one-fourth the number of P3X63 Ag8.6.53 (ATCC CRL 1580) nonsecretingmouse myeloma cells. Cells were plated at approximately 1×10⁵/well inflat bottom microtiter plate, followed by about two week incubation inselective medium containing 10% fetal calf serum, supplemented withorigen (IGEN) in RPMI, L-glutamine, sodium pyruvate, HEPES, penicillin,streptamycin, gentamycin, 1×HAT, and β-mercaptoethanol. After 1-2 weeks,cells were cultured in medium in which the HAT was replaced with HT.Individual wells were then screened by ELISA (described above) for humananti-LAG-3 monoclonal IgG antibodies. Once extensive hybridoma growthoccurred, medium was monitored usually after 10-14 days. The antibodysecreting hybridomas were replated, screened again and, if stillpositive for human IgG, anti-LAG-3 monoclonal antibodies were subclonedat least twice by limiting dilution. The stable subclones were thencultured in vitro to generate small amounts of antibody in tissueculture medium for further characterization.

Hybridoma clones 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5 were selected forfurther analysis and sequencing.

Example 2: Structural Characterization of Human Anti-LAG-3 MonoclonalAntibodies 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5

The cDNA sequences encoding the heavy and light chain variable regionsof the mAbs expressed by the 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5clones, as described in Example 1, were sequenced using the followingprotocol. Total RNA was prepared from 5×10⁶ hybridoma cells using theRNeasy Mini Kit (Qiagen, Valencia, Calif.). cDNA was prepared by the5′-RACE protocol with the SMART RACE cDNA Amplification Kit (ClontechLaboratories, Inc., Mountain View, Calif.) and SuperScript II ReverseTranscriptase (Invitrogen, Carlsbad, Calif.). V-regions of each antibodywere amplified using a 3′ human-specific constant region primer, pairedwith the 5′ RACE universal primer mix. PCR products containing theV-region were cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad,Calif.) and transformed into E. coli strain TOP10 (Invitrogen, Carlsbad,Calif.). Either miniprep DNA or Templiphi (GE Healthcare Biosciences,Piscataway, N.J., USA) samples were prepared, and subjected to DNAsequencing (Sequetech, Mountain View, Calif.). The resultant DNAsequences were analyzed for in-frame rearrangements and other antibodycharacteristics. The expressed proteins were characterized by standardprotein chemistry analysis. The 25E3, 25F7 and 26H10 clones were foundto express an antibody comprising an IgG1 heavy chain and a kappa lightchain, whereas the 8B7 and 17E5 clones were found to express an antibodycomprising an IgG4 heavy chain and a kappa light chain and the 11F2clone was found to express an antibody comprising an IgG2 heavy chainand a kappa light chain.

The nucleotide and amino acid sequences of the heavy chain variableregion of 25F7 are shown in FIG. 1A and in SEQ ID NO: 49 and 37,respectively. The nucleotide and amino acid sequences of the kappa lightchain variable region of 25F7 are shown in FIG. 1B and in SEQ ID NO: 55and 43, respectively. Comparison of the 25F7 heavy chain immunoglobulinsequence to the known human germline immunoglobulin heavy chainsequences (FIG. 7) showed that the 25F7 heavy chain utilizes a V_(H)segment from human germline V_(H) 4-34 (SEQ ID NO:61), and a JH segmentfrom human germline JH5b (SEQ ID NO:62). Further analysis of the 25F7V_(H) sequence using the Kabat system of CDR region determination led tothe delineation of the heavy chain CDR1, CDR2 and CDR3 regions as shownin FIG. 1A and in SEQ ID NOs: 1, 7 and 13, respectively. Comparison ofthe 25F7 light chain immunoglobulin sequence to the known human germlineimmunoglobulin light chain sequences (FIG. 8) showed that the 25F7 kappalight chain utilizes a V_(K) segment from human germline V_(K) L6 (SEQID NO:63) and a J_(K) segment from human germline JK 2 (SEQ ID NO:64).Further analysis of the 25F7 V_(K) sequence using the Kabat system ofCDR region determination led to the delineation of the light chain CDR1,CDR2 and CDR3 regions as shown in FIG. 1B and 1n SEQ ID NOs: 19, 25 and31, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 26H10 are shown in FIG. 2A and in SEQ ID NO: 50 and 38,respectively. The nucleotide and amino acid sequences of the light chainvariable region of 26H10 are shown in FIG. 2B and in SEQ ID NO: 56 and44, respectively. Comparison of the 26H10 heavy chain immunoglobulinsequence to the known human germline immunoglobulin heavy chainsequences (FIG. 9) showed that the 26H10 heavy chain utilizes a V_(H)segment from human germline V_(H) 3-33 (SEQ ID NO:65), and a JH segmentfrom human germline JH 6B (SEQ ID NO:66). Further analysis of the 26H10V_(H) sequence using the Kabat system of CDR region determination led tothe delineation of the heavy chain CDR1, CDR2 and CDR3 regions as shownin FIG. 2A and in SEQ ID NOs: 2, 8 and 14, respectively. Comparison ofthe 26H10 light chain immunoglobulin sequence to the known humangermline immunoglobulin light chain sequences (FIG. 10) showed that the26H10 kappa light chain utilizes a V_(k) segment from human germlineV_(K) A27 (SEQ ID NO:67) and a J_(K) segment from human germline JK 3(SEQ ID NO:68). Further analysis of the 26H10 V_(k) sequence using theKabat system of CDR region determination led to the delineation of thelight chain CDR1, CDR2 and CDR3 regions as shown in FIG. 2B and in SEQID NOs: 20, 26 and 32, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 25E3 are shown in FIG. 3A and in SEQ ID NO: 51 and 39,respectively. The nucleotide and amino acid sequences of the light chainvariable region of 25E3 are shown in FIG. 3B and in SEQ ID NO: 57 and45, respectively. Comparison of the 25E3 heavy chain immunoglobulinsequence to the known human germline immunoglobulin heavy chainsequences (FIG. 11) showed that the 25E3 heavy chain utilizes a V_(H)segment from human germline V_(H) 3-20 (SEQ ID NO:69), and a JH segmentfrom human germline JH 4b (SEQ ID NO:70). Further analysis of the 25e3V_(H) sequence using the Kabat system of CDR region determination led tothe delineation of the heavy chain CDR1, CDR2 and CDR3 regions as shownin FIG. 3A and in SEQ ID NOs: 3, 9 and GGY, respectively. Comparison ofthe 25E3 light chain immunoglobulin sequence to the known human germlineimmunoglobulin light chain sequences (FIG. 12) showed that the 25E3kappa light chain utilizes a V_(k) segment from human germline V_(K) L18(SEQ ID NO:71) and a J_(K) segment from human germline JK 2 (SEQ IDNO:64). Further analysis of the 25E3 V_(k) sequence using the Kabatsystem of CDR region determination led to the delineation of the lightchain CDR1, CDR2 and CDR3 regions as shown in FIG. 3B and in SEQ ID NOs:21, 27 and 33, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 8B7 are shown in FIG. 4A and in SEQ ID NO: 52 and 40,respectively. The nucleotide and amino acid sequences of the light chainvariable region of 8B7 are shown in FIG. 4B and in SEQ ID NO: 58 and 46,respectively. Comparison of the 8B7 heavy chain immunoglobulin sequenceto the known human germline immunoglobulin heavy chain sequences (FIG.13) showed that the 8B7 heavy chain utilizes a V_(H) segment from humangermline V_(H) 4-34 (SEQ ID NO:61), and a JH segment from human germlineJH 5B (SEQ ID NO:62). Further analysis of the 8B7 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 4A and inSEQ ID NOs: 4, 10 and 16, respectively. Comparison of the 8B7 lightchain immunoglobulin sequence (FIG. 14) to the known human germlineimmunoglobulin light chain sequences showed that the 8B7 kappa lightchain utilizes a V_(k) segment from human germline V_(K) L6 (SEQ IDNO:63) and a J_(K) segment from human germline JK 4 (SEQ ID NO:72).Further analysis of the 26H10 V_(k) sequence using the Kabat system ofCDR region determination led to the delineation of the light chain CDR1,CDR2 and CDR3 regions as shown in FIG. 4B and in SEQ ID NOs: 22, 28 &34, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 11F2 are shown in FIG. 5A and in SEQ ID NO: 53 and 41,respectively. The nucleotide and amino acid sequences of the light chainvariable region of 11F2 are shown in FIG. 5B and in SEQ ID NO: 59 and47, respectively. Comparison of the 11F2 heavy chain immunoglobulinsequence to the known human germline immunoglobulin heavy chainsequences (FIG. 15) showed that the 11F2 heavy chain utilizes a V_(H)segment from human germline V_(H) 1-24 (SEQ ID NO:73), a D segment fromthe human germline 2-15, and a JH segment from human germline JH 4B (SEQID NO:70). Further analysis of the 11F2 V_(H) sequence using the Kabatsystem of CDR region determination led to the delineation of the heavychain CDR1, CDR2 and CDR3 regions as shown in FIG. 13A and in SEQ IDNOs: 5, 11 and 17, respectively. Comparison of the 11F2 light chainimmunoglobulin sequence to the known human germline immunoglobulin lightchain sequences (FIG. 16) showed that the 11F2 kappa light chainutilizes a V_(k) segment from human germline V_(K) L6 (SEQ ID NO:63) anda J_(K) segment from human germline JK 1 (SEQ ID NO:74). Furtheranalysis of the 11F2 V_(k) sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCDR3 regions as shown in FIG. 5B and in SEQ ID NOs: 23, 29 and 35,respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 17E5 are shown in FIG. 6A and in SEQ ID NO: 54 and 42,respectively. The nucleotide and amino acid sequences of the light chainvariable region of 17E5 are shown in FIG. 6B and in SEQ ID NO: 60 and48, respectively. Comparison of the 17E5 heavy chain immunoglobulinsequence to the known human germline immunoglobulin heavy chainsequences (FIG. 17) showed that the 17E5 heavy chain utilizes a V_(H)segment from human germline V_(H) 3-33 (SEQ ID NO:65), a D segment fromthe human germline 2-2, and a JH segment from human germline JH 4B (SEQID NO:70). Further analysis of the 17E5 V_(H) sequence using the Kabatsystem of CDR region determination led to the delineation of the heavychain CDR1, CDR2 and CDR3 regions as shown in FIG. 6A and in SEQ ID NOs:6, 12 and 18, respectively. Comparison of the 17E5 light chainimmunoglobulin sequence to the known human germline immunoglobulin lightchain sequences (FIG. 18) showed that the 17E5 kappa light chainutilizes a V_(k) segment from human germline V_(K) L6 (SEQ ID NO:63) anda J_(K) segment from human germline JK 5 (SEQ ID NO:75). Furtheranalysis of the 17E5 V_(k) sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCDR3 regions as shown in FIG. 6B and in SEQ ID NOs: 24, 30 and 36,respectively.

The 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5 variable regions can beconverted to full-length antibodies of any desired isotype usingstandard recombinant DNA techniques. For example, DNA encoding the V_(H)and V_(L) regions can be cloned into an expression vector that carriesthe heavy and light chain constant regions such that the variableregions are operatively linked to the constant regions. Alternatively,separate vectors can be used for expression of the full-length heavychain and the full-length light chain. Non-limiting examples ofexpression vectors suitable for use in creating full-length antibodiesinclude the pIE vectors described in U.S. Patent Publication No.20050153394.

Example 3: Characterization of Binding Properties of LAG-3 MonoclonalAntibodies

In this example, the binding of human anti-LAG-3 antibodies to cellsurface LAG-3 (human, monkey and mouse LAG-3) was examined by flowcytometry. Furthermore, binding kinetics to LAG-3 were analyzed byBIACORE analysis. Still further epitope mapping was conducted using apeptide scan experiment.

A. Flow Cytometry Studies

1. CHO-Human LAG-3 Cell Binding

To test the ability of the antibodies to bind to cell surface LAG-3protein, the antibodies were incubated with a CHO cell line that hadbeen transfected to express human LAG-3 on the cell surface. The 25F7,26H10, 25E3, 8B7, 11F2 and 17E5 monoclonal antibodies were seriallydiluted with cold 1×PFAE buffer (1×PBS+2% FBS, 0.02% sodium azide, 2 mMNa EDTA). For the binding reaction, 50 μl of diluted antibody solutionwas added to a 50 μl cell suspension containing 2×10⁵ cells and themixture was incubated on ice for 30 minutes. The cells were then washedtwo times with 1×PFAE buffer. A 1:100 dilution of FITC-labeled goatanti-human kappa light chain antibody (Bethyl Laboratories, Inc., Cat.#A80-115F) was added and the mixture was incubated for 30 minutes at 4°C., followed by washing twice with cold 1×PFAE buffer. After the finalwash, 150 μl of cold 1×PFAE containing 10 μg/mL propidium iodide (RocheApplied Science, Cat #1_348_639) was added to each solution and analysisof antibody binding was carried out by flow cytometry using aFACScalibur flow cytometer (BD Bioscience).

The results of the flow cytometry analysis are summarized below in Table1, which shows EC₅₀ for binding to CHO-human LAG-3, demonstrating that25F7, 26H10, 25E3, 8B7, 11F2 and 17E5 bind effectively to cell-surfacehuman LAG-3, with 25F7 having approximately a 20 fold lower EC₅₀ than25E3 but approximately equivalent EC₅₀ to that of 8B7 and 26H10. TheEC₅₀ results for 11F2 and 17E5 were in the same range as for 25E3.

TABLE 1 Binding of Anti-LAG-3 Antibodies to CHO Cells Expressing HumanLAG-3 Antibody EC₅₀ (nM) 25F7 0.45-2.52 8B7 1.93-4.44 26H10 1.81-3.6411F2 15.12 25E3  14.9-25.39 17E5 12.3 

2. Activated Human CD4⁺ T Cell Binding

To test the ability of the antibodies to bind to native human LAG-3 onthe surface of activated human T cells, resting CD4⁺ T cells wereisolated from purified peripheral blood mononuclear cells and subjectedto three days of stimulation with a combination of anti-CD3 andanti-CD28 antibodies affixed to polystyrene beads. The 25F7, 8B7 and26H10 monoclonal antibodies were serially diluted with cold 1×PFAEbuffer (1×PBS+2% FBS, 0.02% sodium azide, 2 mM Na EDTA). For the bindingreaction, 50 μl of diluted antibody solution was mixed with 50 μl ofPE-labeled anti-human CD4 (BD Bioscience, Cat #555347). Activated Tcells were processed by the same protocol described above. The analysisof antibody binding was conducted as described above.

The results of the flow cytometry analysis are summarized below in Table2, which shows EC₅₀ for binding to activated human CD4⁺ T cells,demonstrating that all three antibodies bind similarly to cell-surfacehuman LAG-3.

TABLE 2 Binding of Anti-LAG-3 Antibodies to Activated human CD4⁺ T cellsAntibody EC₅₀ (nM) 25F7 0.27-0.45 26H10 0.41-0.84 8B7 0.69-1.80

3. Monkey LAG-3 Antigen Binding

To determine whether the anti-LAG-3 antibodies cross-react with monkeyLAG-3, a cDNA sequence was cloned by RT-PCR from a preparation of pooledcDNA prepared by reverse transcription of RNAs from a collection ofcynomolgus and rhesus monkey tissue samples. The sequence was firstamplified from the cDNA pool using primers (5′ forward primer:5Mcyn1408; 5′-atgtgggaggctcagttcctg-3′ (SEQ ID NO: 91) & 3′ reverseprimer: 3Mcyn1408a; 5′-gtcagagctgctccggctc-3′ (SEQ ID NO: 92)) using aGC-rich PCR amplification system (Roche) and was cloned into a recipientTOPO cloning vector (Invitrogen) for sequence analysis. Clones matchingthe reference Genbank rhesus monkey LAG-3 sequence (Genbank AccessionNo. XM_001108923) were subsequently re-amplified from the TOPO-cloningvector DNA utilizing a second set of primers that incorporatedrestriction enzyme sites for directional cloning in a mammalian cellexpression vector.

Monkey LAG-3 clone pa23-5 was isolated and sequenced. The isolatedmonkey sequence exhibited 99.6% identity to the reference Genbank rhesusmonkey LAG-3 sequence. A comparison of the amino acid sequence of cDNAclone pa23-3 (SEQ ID NO: 93) with rhesus monkey LAG-3 (SEQ ID NO: 94)from Genbank (Accession No. XM_001108923) is shown in FIG. 19. The twosequences are identical except for a one amino acid difference atposition 419 (arginine in clone pa23-5 versus threonine in the Genbankrhesus sequence) and on this basis it is concluded that cDNA clonepa23-5 represents the rhesus LAG-3 gene sequence.

The cDNA of clone pa23-5 was inserted into an expression construct,which was transfected into CHO-S suspension cells by nucleofection(Amaxa). Rhesus LAG-3 expression by sorted, selection drug-resistantclones was verified by FACS analysis. This clonal CHO cell lineover-expressing rhesus LAG-3 was used in similar FACS assays to thosedescribed above to measure antibody cross reactivity to the monkeyprotein. Briefly, the 25F7, 8B7 and 26H10 monoclonal antibodies wereserially diluted with cold 1×PFAE buffer (1×PBS+2% FBS, 0.02% sodiumazide, 2 mM Na EDTA). For the binding reaction, 50 μl of dilutedantibody solution was added to a 50 μl cell suspension containing 2×10⁵cells and the mixture was incubated on ice for 30 minutes. The cellswere processed by the same protocol described above. The analysis ofantibody binding was conducted as described above.

In a separate experiment, the antibodies were tested for binding tocynomolgus monkey LAG-3 using activated cynomolgus monkey T cells. Invitro activation of these monkey T cells was achieved throughanti-CD3/anti-CD28 treatment of the T cells by essentially the sameprotocol described above for the in vitro activation of human T cells,followed by flow cytometry analysis performed as described above forstaining of in vitro activated human CD4⁺ T cells.

The results of the flow cytometry analyses using the CHO-rhesus LAG-3cells and the activated cynomolgus T cells are summarized below in Table3, which shows EC₅₀ for binding to the two different types of cellsexpressing monkey LAG-3. These results showed that all antibodies bindeffectively to both LAG-3 on the activated cynomolgus T cells and therhesus LAG-3 (SEQ ID NO: 93) transfected into CHO cells. There is ahierarchy, however, of binding affinities, with clone 26H10 showing thehighest affinity, which is approximately 2.5 and 6-fold better than thatof clones 8B7 and 25F7, respectively. Difference in binding hierarchybetween the two cell types may reflect amino acid sequence differencesbetween the rhesus and cynomolgus LAG-3 proteins.

TABLE 3 Binding of Anti-LAG-3 Antibodies to Monkey LAG-3 Activated CynoCD4⁺ T CHO-rhesus LAG3 Antibody cells EC₅₀ (nM) EC₅₀ (nM) 26H10 5.194.684 25F7 14.18 22.72 8B7 30.45 10.01

4. Mouse LAG-3 Antigen Binding

To determine whether the antibodies cross-reacted with mouse LAG-3,similar flow cytometry studies to those described above were performedusing as the target cell a mouse T cell hybridoma cell line (3A9) thathad been transfected to express mouse LAG-3 on its cell surface,followed by FACS analysis to detect antibody binding. The resultsindicated that, in contrast to a control anti-mouse LAG3 controlantibody which showed strong staining, none of the human antibodies25E3, 25F7, 8B7 or 26H10 exhibited binding above background levels tocell surface mouse LAG-3, demonstrating that none of these antibodiescross-react with mouse LAG-3.

B. BIACORE Analysis

The binding of the 25E3, 25F7, 8B7, 26H10 and 17E5 antibodies torecombinant LAG-3 protein was examined by BIAcore™ using a capturemethod. The 25E3, 25F7, 8B7, 26H10 and 17E5 antibodies each werecaptured using anti-CH1, a reagent antibody that is specific towards theheavy chain constant region 1 of human antibody (Zymed, Clone HP6045,Stock conc. 1.0 mg/mL). Anti-CH1 was coated on a CM5 chip (BR-1000-14,Research Grade) at high density (9700-11500RUs). The coating was carriedout based on the standard immobilization procedure recommended by themanufacturer. The 25E3, 25F7, 8B7, 26H10 or 17E5 purified antibody, withconcentrations ranging from 0.5-3 μg/mL, was then captured on theanti-CH1 coated surface at the flow-rate of 10 uL/min for 1 minute. Asingle concentration of recombinant human LAG-3 fusion protein (20 nM)was injected over captured antibody for 3 minutes at a flow rate of 25μg/mL. The antigen was allowed to dissociate for 7.5 minutes. The chipsurface was regenerated after each cycle with 25 μL of 25 mM NaOHfollowed by 30 μL of HBS-EP wash. Isotype controls were run on the chip,and the data used to subtract non-specific binding. All the experimentswere carried out on a Biacore 3000 surface plasmon resonance instrument,using BIAcore Control software v 3.2. Data analysis was carried outusing BiaEvaluation v3.2 software. The results are shown in Table 4below. The BIAcore results for 25E3, 25F7, 8B7, 26H10 and 17E5 confirmthe flow cytometry results that all five antibodies are capable ofbinding with high affinity to human LAG-3.

TABLE 4 Binding Kinetics of Anti-LAG-3 Antibody to Recombinant HumanLAG-3 Antibody K_(D) × 10⁻⁹ (M) 25E3 0.09 8B7 0.09 26H10 0.10 25F7 0.4717E5 4.53

C. Epitope Mapping

In the LAG-3 protein, the immunoglobulin-like first domain of theextracellular region contains an exposed “extra loop” having the aminoacid sequence: GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 79). Toexamine the binding of 25E3, 25F7, 8B7 and 26H10 to this region ofLAG-3, and map the epitope bound by each antibody, a peptide scanexperiment was performed across this region. A series of 10 overlappingpeptides that scanned across the full length of the extra loop sequencewere prepared and conjugated to biotin. For ELISA analysis, microtiterplates pre-coated with streptavidin (Sigma-Aldrich, Cat #M5432) wereused to capture the biotinylated loop peptide-conjugates applied in a100 μl volume at a concentration of 2 μg/mL and incubated 18 hours at 4°C., after which the plates were washed 3 times and blocked at roomtemperature for 1 hour with blocking buffer (1×PBS+10% FBS). Next, humananti-LAG-3 antibodies serially diluted 3-fold in blocking buffer from 10μg/mL were applied and the plates were incubated at room temperature for2 hours and then washed three times. To detect bound human antibody aHRP-conjugated goat anti-human kappa light chain antibody (BethylLaboratories, Cat #A80-115P) was diluted to 1 μg/mL in blocking bufferand applied to assay wells for 1 hour followed by three washes andapplication of TMB substrate (eBioscience, Cat #00-4201-56). Opticaldensity readings at 650 nm wavelength were made on a Spectramax 340PCspectrophotometer (Molecular Dynamics, Inc.). The results of the peptidescan experiment are summarized below in Table 5.

TABLE 5 Anti-LAG Antibody Binding to Peptide Scan of LAG-3 Extra LoopLAG-3 Extra Loop Peptide Scan SEQ GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY 79 25E38B7 25F7 26H10 GPPAAAPGHPLA 80 - - - -   PAAAPGHPLAPG 81 ++ - - -    AAPGHPLAPGPH 82 ++ - - -       PGHPLAPGPHPA 83 + - - -        HPLAPGPHPAAP 84 ± - - -           LAPGPHPAAPSS 85 - - - -            PGPHPAAPSSWG 86 - ++ ++ -               PHPAAPSSWGPR 87 - ++++ -                 PAAPSSWGPRPR 88 - ++ + -                  APSSWGPRPRRY 89 - - - -Based on these results, it was determined that the 25E3 antibodyrecognized a region within the extracellular loop comprising the aminoacid sequence PGHPLAPG (SEQ ID NO: 76), whereas the 25F7 antibodyrecognized a region within the extra loop comprising the amino acidsequence HPAAPSSW (SEQ ID NO: 77) and 8B7 appeared to recognize a regionwithin the extracellular loop comprising the amino acid sequencePAAPSSWG (SEQ ID NO: 78). In contrast, no binding of the full lengthextra loop peptide or any of the shorter scanning peptides by the 26H10antibody could be detected.

The regions identified in this study are underlined in the full-lengthextra loop sequence:

Thus, the peptide scan results indicate that the 25E3, 25F7 and 8B7antibodies bind to different although closely located epitopes withinhuman LAG-3.

To further examine binding of these antibodies to the extra loop peptideregion, additional ELISA assays were performed. In an ELISA assay usingthe human full-length extra loop peptide (SEQ ID NO: 79), EC₅₀ valuesfor binding were determined for 25E3, 25F7 and 8B7. Additionally, asimilar peptide ELISA was conducted using the full length extra looppeptide sequence from rhesus monkey LAG-3, having the sequenceGPPAPAPGHPPAPGHRPAAP YSWGPRPRRY (SEQ ID NO: 90), and EC₅₀ values forbinding were determined for 25F7 and 8B7. The results are summarizedbelow in Table 6. The results confirm that antibodies 25E3, 25F7 and 8B7are capable of recognizing the human LAG-3 extra loop peptide region.Moreover, antibodies 25F7 and 8B7 also bind to the rhesus LAG-3 extraloop peptide region, albeit less well compared to the human sequence,which may be due to the species sequence divergence in this polypeptide.The results also confirm that the 26H10 antibody is not capable ofrecognizing the LAG-3 extra loop peptide.

TABLE 6 Binding of Anti-LAG-3 Antibodies to Human and Rhesus LAG-3 ExtraLoop Peptide Human Extra Loop Rhesus Extra Loop Antibody EC₅₀ (nM) EC₅₀(nM) 25E3 0.55 Not tested 25F7 0.29-0.95 13.09 8B7 0.28-1.35  0.60 26H10No binding No binding

Example 4: Inhibition of Binding of LAG-3 to MHC Class II by Anti-LAG-3mAbs

To test the ability of the anti-LAG-3 antibodies to inhibit binding ofLAG-3 to MHC Class II molecules, an in vitro binding assay was performedin which a LAG-3 fusion protein, comprising human LAG-3 extracellardomain fused to mouse Fc (hLAG-3-mIg), was reacted with Daudi cells,which express human MHC Class II molecules.

To test antibody inhibition of LAG-3 binding to MHC Class II, 25E3,25F7, 8B7 and 26H10 were serially diluted from 20 μg/mL in PFAE bufferand to these serial dilutions was added 1 μg/ml of hLAG-3-mIg fusionprotein. This mixture was incubated for 20 minutes at room temperatureprior to adding to 2×10⁵ 1×PFAE-washed Daudi cells. The mixture wasapplied to Daudi cells and incubated at 4° C. for 30 minutes. The cellswere pelleted (three minutes, 400×g), washed once with 1×PFAE buffer andre-pelleted, and binding of hLAG-3-mIg to the Daudi cells was detectedusing a recombinant PE-labeled anti-mIgG Fcγ secondary reagent. Analysisof LAG-3-mIg binding was carried out with the FACScalibur flow cytometer(BD Bioscience). The results are summarized in Table 7 below, whichshows IC₅₀ values in nM.

TABLE 7 Inhibition of LAG-3 Binding to MHC Class II by Anti-LAG-3Antibodies Antibody IC₅₀ (nM) 25E3  0.8-6.78 25F7 0.12-0.92 8B70.19-0.95 26H10 0.10The results demonstrate that all four antibodies are effective ininhibiting binding of LAG-3 to MHC Class II antibodies, with 25F7, 8B7and 26H10 exhibiting IC₅₀ values approximately 7 to 13-fold lower thanthat of 25E3.

Example 5: Stimulation of Antigen-Specific T Cell Response by Anti-LAG-3mAbs

To test the ability of the anti-LAG-3 antibodies to stimulate anantigen-specific T cell response, a 3A9 T Cell Peptide Stimulation Assay(see e.g., Workman et al. (2003) J. Immunol. 169:5392-5395; Workman etal. (2002) Eur. J. Immunol. 32:2255-2263) was used.

In this assay, a mouse T cell hybridoma, 3A9, specific for the peptideHEL₄₈₋₆₂, was used as the responder T cell. The responder 3A9 T cell wasretrovirally transduced to express either human LAG-3 or mouse LAG-3 onits cell surface. The antigen presenting cell (APC) used to present theHEL₄₈₋₆₂ peptide antigen to the 3A9 cells was the mouse MHC Class IIpositive cell line LK35.2. Separate studies determined that a humanLAG-3 fusion protein was capable of binding to mouse MHC Class IImolecules, thereby validating the use of LK35.2 mouse APCs in thisassay. Antigen-specific stimulation of the 3A9 cells was indicated byproduction of interleukin-2 (IL-2), the secretion of which was measuredby ELISA (mouse IL-2 OptEIA kit, BD Bioscience, Cat #555148 according tomanufacturer's recommendations).

Ectopic expression of human or mouse LAG-3 on the 3A9 T cells, in theabsence of any antibodies, led to an inhibitory effect onantigen-specific responses when the transfected T cells were incubatedwith the LK35.2 APCs presenting the HEL₄₈₋₆₂ peptide antigen, asindicated by an increase in the amount of peptide antigen needed tostimulate IL-2 production by the 3A9 cells in comparison to the peptidedose response profile of control 3A9 T cells.

To test antibody stimulation of the antigen-specific T cell response,the APC (2.5×10⁴ cells) was first preincubated with the antigenicpeptide (200 nM) for 30 minutes at 37° C. and the 3A9 T cells (5.0×10⁴cells expressing either mLAG-3, hLAG-3 or control cells) werepreincubated with an anti-hLAG-3 antibody (25E3, 25F7, 8B7, 26H10, 11F2,17E5), serially diluted in three fold dilution from 25 μg/mL) for 15minutes at 37° C. The 3A9 T cells were then added to the antigen-pulsedAPCs and the culture incubated for 24 hours at 37° C. The supernatantswere then harvested and measured for production of mouse IL-2. Theresults for the 3A9 T cells expressing human LAG-3 are in Table 8, whichshows IC₅₀ values in nM.

TABLE 8 Stimulation of Antigen-Specific T Cell Responses by Anti-LAG-3Antibodies Antibody 3A9-hLAG-3 Peptide Assay IC₅₀ (nM) 25F7 0.14-1.9426H10 1.45-6.49 8B7  3.25-13.90 25E3  3.88-70.78 11F2 81.50-240   17E5No inhibitionThe results show that antibodies 25F7, 8B7 and 26H10, and to a lesserextent 25E3, were able to stimulate IL-2 production in anantigen-specific T cell response assay, whereas antibody 11F2 exhibitedminimal ability to inhibit and antibody 17E5 was not functional in thisassay. None of the antibodies altered the measured IL-2 production bycontrol 3A9 T cells or 3A9 T cells transfected with mouse LAG-3 protein,demonstrating the specificity of the stimulatory effect.

Example 6: Tumor Growth Inhibition by Anti-LAG-3 mAb, Alone or inCombination

To test the ability of anti-LAG-3 antibody, alone or in combination withanother immunostimulatory antibody, to inhibit the growth of tumor cellsin vivo, two different syngeneic mouse tumor graft models were used. Thefirst model used murine SalN fibrosarcoma cells. The second model usedthe murine MC38 colon cancer cell line.

In a first experiment, mice (A/J strain) were each implanted with 2×10⁶SalN fibrosarcoma cells on day 0 and the tumor cells were allowed togrow for seven days. On day 7, day 10 and day 12 post-implantation, themice were treated with 10 mg/kg of either an anti-LAG-3 mAb alone (therat anti-mouse LAG-3 mAb C9B7W; eBioscience, Cat. No. 14-2231), ananti-PD-L1 antibody alone (an anti-mouse PD-L1 mAb 14D8), the anti-LAG-3and anti-PD-L1 antibodies in combination, or an IgG1 isotype controlantibody. The 14D8 mAb is a rat anti-mouse PD-L1 antibody that has beenchimerized to contain the mouse IgG1 and mouse kappa constant regions.

Tumor volumes in the mice were measured for over 50 dayspost-implantation and mean and median tumor volumes were determined.Mean tumor growth inhibition was calculated (based on treatment with theisotype control IgG1 antibody being 0% inhibition). The results for day24 post-implantation are summarized below in Table 9:

TABLE 9 Mean Tumor Growth Inhibition in Sa1N Tumor Model Day IgG1 LAG-3PD-L1 Combo 24 — 68 74.9 95.8Thus, anti-LAG3 antibody alone, or anti-PD-L1 antibody treatment alone,resulted in tumor growth inhibition, while the combination of bothantibodies led to even greater tumor growth inhibition. With respect tothe treatment groups, by the end of the experiment the results were that4 of 10 mice treated with anti-LAG3 alone became tumor free, whereasonly 1 of 10 mice treated with the control IgG1 antibody became tumorfree. Similarly, 4 of 11 mice treated with anti-PD-L1 alone wererendered tumor free. Treatment of mice with the combination of anti-LAG3and anti-PD-L1 resulted in 9 of 10 mice becoming tumor free; theremaining mouse not tumor free had an indolent tumor that remained smallthroughout the study.

Two additional studies used mice implanted with cells of the murine MC38colon cancer cell line. In the first experiment, C57Bl/6 mice were eachimplanted with 2×10⁶ MC38 cells on day 0, and were treated on day 7, day10 and day 12 post-implantation with 200 μg/dose of anti-LAG-3 alone(C9B7W mAb), anti-PD-1 alone (the 4H2 mAb) or anti-LAG-3 and anti-PD-1in combination. An IgG1 isotype matched antibody, at 400 μg/dose, wasused as a control. The 4H2 mAb is a rat anti-mouse PD-1 antibody thathas been chimerized to contain the mouse IgG1 and mouse kappa constantregions.

Mean tumor volume, median tumor volume and % survival was determined at80 days post-implantation. The results showed that LAG-3 monotherapy inthis tumor model (MC38) showed little or no activity in inhibiting tumorgrowth and none of the treated mice survived the duration of theexperiment. In contrast, anti-PD-1 monotherapy showed significantactivity, with 4 of 10 mice tumor free at the end of the experiment.Moreover, similar to the results with the SalN model, the combinationtherapy of anti-LAG-3 plus anti-PD-1 was more effective than eithertreatment alone, with 7 of 8 mice being tumor free at the end of theexperiment.

In a second experiment with the MC38 model, C57Bl/6 mice were eachimplanted with 2×10⁶ MC38 cells on day 0, and were treated on day 5, day8 and day 11 post-implantation with 200 μg/dose of test antibody and/or400 μg/dose control IgG antibody, as follows: (i) an anti-IgG1 controlantibody; (ii) an anti-LAG-3 mAb (C9B7W mAb) together with the controlIgG1; (iii) an anti-PD-1 antibody (4H2) together with the control IgG1;(iv) an anti-CTLA-4 antibody (the 9D9 mouse anti-mouse CTLA-4 mAb)together with the control IgG1; (v) the anti-LAG-3 mAb together with theanti-PD-1 mAb; or (vi) the anti-LAG-3 mAb together with the anti-CTLA-4mAb. The 9D9 mAb is a mouse anti-mouse CTLA-4 antibody that was raisedin a mouse in which the endogenous mouse CTLA-4 had been knocked out.

Mean tumor volume, median tumor volume and % survival was determined forover 100 days post-implantation. The results were similar to the firstexperiment in that LAG-3 monotherapy showed little or no activity ininhibiting MC38 tumor growth and none of the treated mice survived theduration of the experiment. CTLA-4 monotherapy also showed little or noactivity in inhibiting MC38 tumor growth and none of the treated micesurvived the duration of the experiment. In contrast, anti-PD-1monotherapy again showed significant activity, with 4 of 10 mice tumorfree at the end of the experiment. Moreover, again combination therapywas more effective than monotherapy. For mice treated with thecombination of anti-LAG-3 and anti-CTLA-4, 3 of 10 mice were tumor freeat the end of the experiment and for the mice treated with thecombination of anti-LAG-3 and anti-PD-1, 8 of 10 mice were tumor free atthe end of the experiment.

Thus, the above-described in vivo tumor graft studies demonstrated that,for at least certain tumor models, anti-LAG antibody treatment aloneresulted in significant inhibition of tumor growth in vivo. Furthermore,for multiple tumor models, the combination therapy of anti-LAG-3antibody together with either anti-PD-1 antibody, anti-PD-L1 antibody oranti-CTLA-4 antibody resulted in even greater anti-tumor activity thanmonotherapy alone.

Example 7: Promotion of Autoimmunity in NOD Mice by Inhibition byAnti-LAG-3 mAb

To test the ability of anti-LAG-3 antibody to stimulate an immuneresponse, as indicated by the development of autoimmunity, the NOD mousemodel of diabetes was utilized. NOD mice are known to be prone todeveloping autoimmune diabetes. Progression of diabetes can be followedin female NOD mice by measuring serum glucose. Thus, the effect ofanti-LAG-3 treatment, alone or in combination with eitherimmunostimulatory antibodies, on the development of diabetes in femaleNOD mice was examined.

Female NOD mice were treated on day 0, day 2 and day 5 with 250 μg/doseof either: (i) an IgG1 isotype control antibody; (ii) anti-LAG-3 mAbalone (C9B7W mAb); (iii) anti-PD-1 mAb alone (4H2 mAb); (iv) anti-CTLA-4mAb alone (9D9 mAb); (v) anti-LAG-3 mAb together with anti-PD-1 mAb; or(vi) anti-LAG-3 mAb together with anti-CTLA-4. The results demonstratedwith anti-LAG-3 treatment alone or anti-PD-1 treatment alone (but notanti-CTLA-4 treatment alone) increased the number of mice converting tothe diabetic phenotype. Moreover, the combination treatment ofanti-LAG-3 plus anti-PD-1, or anti-LAG-3 plus anti-CTLA-4, was even moreeffective in converting mice to the diabetic phenotype.

Thus, these results demonstrate that blockade of LAG-3 interaction withits receptor interfered with a negative immunoregulatory signal thatallowed for greater immunological activity in the NOD mice, and thisgreater immunological activity in the LAG-3 treated mice could beenhanced by combination treatment with either anti-PD-1 or anti-CTLA-4antibody.

Example 8: Immunohistochemistry Using Anti-LAG-3 mAbs

In this experiment, fluorescently-labeled anti-LAG-3 human antibodieswere used in immunohistochemistry experiments. The followingFITC-labeled, human anti-LAG-3 antibodies were used: 25F7-FITC (F:P=2.9;IgG1 version); 25F7-G4-FITC (F:P=2.7; IgG4 version); 8B7-FITC (F:P=2.6)and 26H10-FITC (F:P=3.4). A panel of lymphoid tissues, specificallytonsil (two samples), spleen (two samples) and thymus (two samples), wasexamined, along with pituitary tissue (four samples). LAG-3 transfectedCHO cells also were used as a control. Acetone-fixed cryostat sectionswere used. The sections were stained with FITC-labeled anti-LAG-3antibody (0.2-5 μg/ml), followed by staining with a rabbit anti-FITCantibody as a bridge antibody and then visualization using the rabbitEnVision™+ System Kit (Dako USA, Carpinteria, Calif.). The results aresummarized below in Table 10.

TABLE 10 Immunohistochemistry using Anti-LAG-3 mAbs Tissue 25F7-FITC25F7-G4-FITC 8B7-FITC 26H10-FITC CHO/LAG-3 + + + + Cells (strong)(strong) (strong) (strong) Tonsil + + + + (n = 2) (strong; rare in(strong; rare in (strong; rare in (strong; rare in scattered LC,scattered LC, scattered LC, scattered LC, 2/2) 2/2) 2/2) 2/2)Spleen + + + + (n = 2) (very weak, (very weak, (weak, mainly in (veryweak, mainly in red mainly in red red pulp, 2/2) mainly in red pulp,2/2) pulp, 2/2) pulp, 2/2) Thymus + + + + (n = 2) (strong; very rare(strong; very rare (strong; very (strong; very rare in scattered LC, inscattered LC, rare in scattered in scattered LC, 1/2) 1/2) LC, 1/2) 1/2)Pituitary + + − + (n = 4) (strong; (strong; (strong; occasional inoccasional in occasional in adeno- adeno- adeno- hypophysis, 3/4)hypophysis, 3/4) hypophysis, 3/4; weak moderate, rare, 1/4) LC =lymphocyte; + = positive staining; − = negative stainingAs expected, LAG-3 expression was detected in the panel of lymphoidtissue. Additionally, two of the three anti-LAG-3 antibodies examined,25F7 (IgG1 and IgG4 versions) and 26H10, exhibited retention inpituitary tissue, whereas one antibody examined, 8B7, did not show thisretention in the pituitary tissue. Thus, the immunohistochemistryexperiment identified two subsets of anti-LAG-3 antibodies, wherein onesubset is retained in pituitary tissue and the other subset is notretained in pituitary tissue.

SUMMARY OF SEQUENCE LISTING

SUMMARY OF SEQUENCE LISTING SEQ ID NO: SEQUENCE SEQ ID NO: SEQUENCE  1V_(H) CDR1 a.a. 25F7 49 V_(H) n.t. 25F7  2 V_(H) CDR1 a.a. 26H10 50V_(H) n.t. 26H10  3 V_(H) CDR1 a.a. 25E3 51 V_(H) n.t. 25E3  4V_(H) CDR1 a.a. 8B7 52 V_(H) n.t. 8B7  5 V_(H) CDR1 a.a. 11F2 53V_(H) n.t. 11F2  6 V_(H) CDR1 a.a. 17E5 54 V_(H) n.t. 17E5  7V_(H) CDR2 a.a. 25F7 55 V_(K) n.t. 25F7  8 V_(H) CDR2 a.a. 26H10 56V_(K) n.t. 26H10  9 V_(H) CDR2 a.a. 25E3 57 V_(K) n.t. 25E3 10V_(H) CDR2 a.a. 8B7 58 V_(K) n.t. 8B7 11 V_(H) CDR2 a.a. 11F2 59V_(K) n.t. 11F2 12 V_(H) CDR2 a.a. 17E5 60 V_(K) n.t. 17E5 13V_(H) CDR3 a.a. 25F7 61 V_(H) 4-34 germline a.a. 14V_(H) CDR3 a.a. 26H10 62 V_(H) JH5b germline a.a. 15 PVGVV 63V_(k) L6 germline a.a. 16 V_(H) CDR3 a.a. 8B7 64 V_(k) JK2 germline a.a.17 V_(H) CDR3 a.a. 11F2 65 V_(H) 3-33 germline a.a. 18V_(H) CDR3 a.a. 17E5 66 V_(H) JH6b germline a.a. 67V_(k) A 27 germline a.a. 19 V_(K) CDR1 a.a. 25F7 68V_(k) JK3 germline a.a. 20 V_(K) CDR1 a.a. 26H10 69V_(H) 3-20 germline a.a. 21 V_(K) CDR1 a.a. 25E3 22 V_(K) CDR1 a.a. 8B770 V_(H) JH4b germline a.a. 23 V_(K) CDR1 a.a. 11F2 71V_(k) L-18 germline a.a. 24 V_(K) CDR1 a.a. 17E5 72V_(k) JK4 germline a.a. 73 V_(H) 1-24 germline a.a. 25V_(K) CDR2 a.a. 25F7 74 V_(k) JK1 germline a.a. 26 V_(K) CDR2 a.a. 26H1075 V_(k) JK5 germline a.a. 27 V_(K) CDR2 a.a. 25E3 28V_(K) CDR2 a.a. 8B7 76 PGHPLAPG 29 V_(K) CDR2 a.a. 11F2 77 HPAAPSSW 30V_(K) CDR2 a.a. 17E5 78 PAAPSSWG 79 GPPAAAPGHPLAPGPHPAAPSSW GPRPRRY 31V_(K) CDR3 a.a. 25F7 80 GPPAAAPGHPLA 32 V_(K) CDR3 a.a. 26H10 81PAAAPGHPLAPG 33 V_(K) CDR3 a.a. 25E3 82 AAPGHPLAPGPH 34V_(K) CDR3 a.a. 8B7 83 PGHPLAPGPHPA 35 V_(K) CDR3 a.a. 11F2 84HPLAPGPHPAAP 36 V_(K) CDR3 a.a. 17E5 85 LAPGPHPAAPSS 86 PGPHPAAPSSWG 37V_(H) a.a. 25F7 87 PHPAAPSSWGPR 38 V_(H) a.a. 26H10 88 PAAPSSWGPRPR 39V_(H) a.a. 25E3 89 APSSWGPRPRRY 40 V_(H) a.a. 8B7 90 GPPAPAPGHPPAPGHRPAAPYSWGPRPRRY 41 V_(H) a.a. 11F2 42 V_(H) a.a. 17E5 91atgtgggaggctcagttcctg 92 gtcagagctgctccggctc 43 V_(K) a.a. 25F7 93Rhesus LAG-3 clone pa23-5 a.a. 44 V_(K) a.a. 26H10 94 Rhesus LAG-3 a.a.(XM_001108923) 45 V_(K) a.a. 25E3 46 V_(K) a.a. 8B7 47 V_(K) a.a. 11F248 V_(K) a.a. 17E5

What is claimed:
 1. A method for treating cancer in a subject comprisingadministering to the subject an anti-LAG-3 antibody and an anti-PD-L1antibody.
 2. The method of claim 1, wherein the anti-LAG-3 antibodyinhibits binding of LAG-3 to major histocompatibility (MHC) class IImolecules.
 3. The method of claim 1, wherein the anti-LAG-3 antibodystimulates interleukin-2 (IL-2) production in an antigen-specific T cellresponse.
 4. The method of claim 1, wherein the anti-LAG-3 antibody is ahuman antibody.
 5. The method of claim 1, wherein the anti-LAG-3antibody is a chimeric or humanized antibody.
 6. The method of claim 1,wherein the anti-LAG-3 antibody and the anti-PD-L1 antibody arecomprised in a bispecific molecule.
 7. The method of claim 1, whereinthe anti-PD-L1 antibody is a human antibody.
 8. The method of claim 1,wherein the anti-PD-L1 antibody is a chimeric or humanized antibody. 9.The method of claim 1, wherein the anti-LAG-3 antibody and theanti-PD-L1 antibody are administered concurrently as separatecompositions with each antibody in a pharmaceutically acceptablecarrier.
 10. The method of claim 1, wherein the anti-LAG-3 antibody andthe anti-PD-L1 antibody are administered concurrently in the samecomposition.
 11. The method of claim 1, wherein the anti-LAG-3 antibodyand the anti-PD-L1 antibody are administered sequentially.
 12. Themethod of claim 1, further comprising administering a chemotherapeuticagent.
 13. The method of claim 1, further comprising administering anangiogenesis inhibitor.
 14. The method of claim 1, wherein the subjectis a human.
 15. A method for inhibiting growth of tumor cells in asubject comprising administering to the subject an anti-LAG-3 antibodyand an anti-PD-L1 antibody.
 16. The method of claim 15, wherein theanti-LAG-3 antibody is a human antibody.
 17. The method of claim 15,wherein the anti-PD-L1 antibody is a human antibody.
 18. The method ofclaim 15, further comprising administering a chemotherapeutic agent. 19.The method of claim 15, further comprising administering an angiogenesisinhibitor.
 20. The method of claim 15, wherein the subject is a human.